Senator Robert Menendez of New Jersey pleaded not guilty on Monday to a new federal charge that accused him of illegally plotting to be an agent of Egypt while serving as chairman of the Senate Foreign Relations Committee.

It was Mr. Menendez’s second not-guilty plea in a month after he and his wife, Nadine Menendez, were accused of being at the center of a broad web of political corruption. The couple has been charged with accepting bribes in exchange for Mr. Menendez’s efforts to increase aid and weapons sales to Egypt while also working to quash criminal investigations for associates in New Jersey.

The bribery-related charges were first announced last month by federal prosecutors in Manhattan; a revised indictment, made public on Oct. 12, included the new charge of conspiring to act as an agent of a foreign government.

Ms. Menendez, 56, pleaded not guilty to the new conspiracy charge last week, but Mr. Menendez was granted permission to appear on Monday, instead, so that he could be present in Washington for legislative business.

His appearance on Monday took less than five minutes, and he left the courthouse in Lower Manhattan without addressing questions from reporters.

Three New Jersey businessmen, including Wael Hana, a U.S. citizen born in Egypt who founded a halal certification company based in Edgewater, N.J., have also pleaded not guilty to participating in the corruption scheme.

Investigators found bars of gold bullion and $550,000 in cash during searches of the Menendezes’ home in Englewood Cliffs, N.J., and of a safe deposit box. Prosecutors have asked a judge to seize their home and a Mercedes-Benz convertible that the government says was given to them as a bribe.

Last week, Judge Sidney H. Stein refused to permit Mr. Hana to remove the electronic monitor he is required to wear as a condition of his release on a $5 million bond.

Judge Stein called the request premature, noting Mr. Hana’s $25 million in assets and his extensive connections to the Egyptian government, and, specifically, to Egyptian intelligence officers.

“So while we hope that Mr. Hana does not intend to flee the United States,” Judge Stein said, “were he to try to do so, common sense alone would tell you he might well have the assistance of folks to get him a passport, so the mere surrender of his travel documents is not sufficient.”

Mr. Menendez, a Democrat who until last month was the chairman of the powerful Senate Foreign Relations Committee, has maintained his innocence and remained defiant in the face of the federal charges, ignoring dozens of calls from onetime allies who have urged him to resign.

It is the second time in a decade that Mr. Menendez, 69, has been accused of accepting bribes, and he has said that he believed he was being targeted by overzealous prosecutors because he is Latino.

“The government is engaged in primitive hunting,” Mr. Menendez told news reporters last week in Washington. “That’s where you continue to hound your prey until it’s exhausted and then you kill it.

“That’s not going to work with me,” he added. “I have nothing to resign because I’m innocent.”

Judge Stein has set May 6 as a trial date; defense lawyers and prosecutors agreed last week to pause the speedy trial clock until then to provide enough time to review the roughly 7.7 million pages of discovery records collected from 50 electronic devices analyzed during the investigation.

Paul M. Monteleoni, an assistant U.S. attorney in Manhattan, said that would give the parties time to review the extensive discovery material, file motions, prepare for trial “and potentially to discuss the disposition of the case.”

On Monday, a coalition of 25 organizations, including Citizens for Responsibility and Ethics in Washington, Center for American Progress, Project on Government Oversight, and Public Citizen, sent a letter to Mr. Menendez requesting that he resign.

“These allegations paint a damning picture of a senator who seemingly repeatedly placed his own personal interests above the United States’ national security and democratic process,” the letter stated.

But the senator, who had been planning to run for re-election to a fourth full term next year, has given no indication that he intended to comply with that request. A fund-raiser he held last weekend at an exclusive beachfront resort in Puerto Rico drew few donors, but he has roughly $8.6 million in campaign cash, which could be used to pay legal costs. A separate defense fund he set up over the summer has raised $275,000 from about 30 individuals and organizations, Senate records show.

The fund returned a $5,000 donation last month from a New Jersey law firm run by Donald Scarinci, a longtime friend of Mr. Menendez’s. Mr. Scarinci said he was unsure why the donation was rejected, but indicated that it might have been because it came from his company, not his private bank account.

“Bob has been my personal friend for a lifetime,” Mr. Scarinci said in an email. “As his friend, I am there for him in his time of need. I fully intend to contribute to his defense fund.”

Kayla Guo contributed reporting from Washington.