The essential job-saving measure, which passed by unanimous consent after more than a week of negotiations between Democrats, Republicans and the White House, now heads to the House of Representatives where a vote could occur as early as Thursday.
The effort is the government's latest massive cash infusion to prop up a collapsing economy amid struggles to contain a pandemic that has killed 43,000 Americans and left some 22 million people jobless.
"This is a significant package," Senate Majority Leader Mitch McConnell said, one which was cobbled together "on a crash timeline in these most unusual circumstances."
The measure builds on the $2.2 trillion rescue legislation -- the largest-ever federal intervention in the US economy -- signed into law last month.
US President Donald Trump called on Congress to swiftly send him the bill, which would re-fund a program that has already helped more than 1.6 million businesses, so he can sign it into law.
"I urge the House to pass the bill, and they'll be voting on it, I imagine, very soon," he said, AFP reported.
Late last month, with many shops and restaurants nationwide forced to shutter due to the pandemic, the government launched the PPP to provide $350 billion in what were essentially grants as long as businesses use the funds to pay their workers.
Banks were flooded with requests, and the money quickly ran out.
The new tranche would include $320 billion in small business funding, plus $75 billion for hospitals and $25 billion to expand coronavirus testing, McConnell said.
It also provides $60 billion in disaster recovery loans and grants, he added.
"We have taken a giant step forward" towards providing more relief for small businesses and for first responders in hospitals, top Senate Democrat Chuck Schumer said.
Schumer claimed Democratic victory for obtaining funding for testing, and he praised Trump, Treasury Secretary Steven Mnuchin and chief of staff Mark Meadows for agreeing to include it.
"We will not get America moving again until we have a national, well thought out, well executed testing program -- and this administration doesn't have one," Schumer said.
Republicans wanted to pass a measure to fund just the Paycheck Protection Program for small businesses, but ultimately testing and hospital funding was included.
Schumer said considerable PPP money will be reserved for minority-run small businesses and those without access to large financial institutions.
The deal leaves out a key Democratic demand: extra funding for state and local governments battling the pandemic.
Trump said he wants such funding included in the next coronavirus rescue package, and congressional leaders said the forthcoming bill was already in the works.
The next measure will be "bigger, bolder, better" than the interim deal, Schumer said.
With the 100-member Senate having postponed its full sessions until May 4 due to coronavirus, Tuesday's vote occurred during a pro forma session, which usually features few lawmakers but can pass legislation if there is unanimous consent.
The measure passed, but McConnell made clear any subsequent aid package, including funding for state and local governments, would only be considered "when the Senate is back in session, with full participation" in Washington.
A vote in the House, also on recess, this week could be complicated.
The House is unlikely to pass the job-saving package by unanimous consent as the Senate did.
Number two Democrat Steny Hoyer said it likely will have to convene a House session where more than half the members return to Washington to attend.