Democratic Senator Barbara Mikulski announced her support for the agreement, becoming the 32nd Senate Democrat, along with two independents, to back a pact announced on July 14, which exchanges sanctions relief for Iran for Tehran's agreeing to curtail its nuclear program.
The move means Obama's fellow Democrats will have enough votes to protect the nuclear deal between Iran and world powers in the U.S. Congress.
Their next goal is to see if they can gather at least 41 votes in the Senate to use the filibuster procedural rule to block a disapproval resolution in the Senate and keep Obama from having to use his veto power.
"No deal is perfect, especially one negotiated with the Iranian regime. I have concluded that this Joint Comprehensive Plan of Action is the best option available to block Iran from having a nuclear bomb," Mikulski said in a statement.
"For these reasons, I will vote in favor of this deal."
Secretary of State John Kerry gave a speech about the deal in Philadelphia on Wednesday, arguing that its benefits outweigh any drawbacks.
U.S. lawmakers have until Sept. 17 to vote on a "resolution of disapproval," which would weaken the international pact by eliminating Obama's ability to temporarily waive many U.S. sanctions on Iran.
The Senate and House of Representatives are expected to take up the issue as soon as they return to Washington on Sept. 8 after their August recess.
With Republicans virtually united in opposition, Democrats have spent the summer rallying support for an agreement seen as a potential legacy foreign policy achievement for the president.
Deal supporters' first goal was to muster enough votes in the Senate or House to sustain Obama's veto of a disapproval resolution.
Senate leadership aides on both sides of the issue said it was still too early to say whether supporters would be able to secure the next target of 41 votes.
Republicans hold majorities in both the House and Senate, and are likely to pass a Republican-sponsored disapproval resolution. Opponents would have needed two-thirds majorities in both chambers - 34 votes in the Senate or 146 in the House - to override Obama's promised veto.
'FAR FROM BEING IMPLEMENTED'
Mikulski's support leaves 10 undecided Senate Democrats. Two, Charles Schumer and Robert Menendez, have announced that they will oppose it.
So far, at least 93 members of the House, all Democrats, have said they support the bill. Nancy Pelosi, the House Democratic Leader, has said she expects to gather enough support to sustain a veto.
The issue has also begun to play a role in the 2016 U.S. presidential election. All of the main candidates for the Republican nomination oppose the deal, although they differ on how they would try to stop it if they reached the White House.
While strong majorities of Republicans in both the Senate and House have already come out against the accord, it is still possible that one or two congressional Republicans might vote for it.
Congressional Republicans dismissed Wednesday's news, and made clear they still intend to fight the deal.
"Forcing a bad deal, over the objections of the American people and a majority in Congress, is no win for President Obama," said Cory Fritz, a spokesman for John Boehner, the Republican Speaker of the House.
"The White House may have convinced just enough Democrats to back an agreement that legitimizes Iran’s nuclear program, trusts the regime to self-inspect and offers amnesty to terrorists, but this deal is far from being implemented," he said.
Senate Republicans have said they will pursue additional measures to crack down on Iran even if the deal survives congressional review, including imposing additional sanctions over human rights violations or providing financial support to terrorists.
Opponents of the Iran nuclear agreement say that it gives Iran too much sanctions relief in exchange for an insufficient regime for inspecting Iran's nuclear facilities. They worry that Iran will use a $50 billion "windfall" to finance militant groups that might threaten U.S. allies, including Israel.
Israeli Prime Minister Benjamin Netanyahu has been a vociferous opponent of the nuclear agreement, and some pro-Israel groups have spent millions of dollars on advertising campaigns urging U.S. lawmakers to oppose it.