Employees of the Dunav Agregati river shipping company anchored ships and barges in the middle of the river near the city of Novi Sad, some 70 kilometres (50 miles) north of the capital Belgrade, prompting the authorities to halt navigation.
"We haven't received wages and health benefits for over a year, we want them paid in full and with this (protest) we want to draw public attention," said Jozef Pap, a protest leader.
"We will stay here until our demands are met."
The Danube, a major European shipping lane, connects Germany and Austria with Slovakia, Hungary, Croatia, Serbia, Romania and the Black Sea, and is vital for their economies.
Rising social discontent in Serbia threatens to damage the coalition government led by President Boris Tadic's pro-Western Democratic Party as it heads into a parliamentary election on May 6. The Democratic Party is trailing in polls behind the opposition populist Serbian Progressive Party.
The European Union candidate country is struggling with the effects on its economy and public finances of the prolonged debt crisis in the euro zone, Serbia's main trading partner, and with rampant corruption and organised crime.
Last month, hundreds of farmers blocked main roads in the north of the country in protest at a new programme of subsidies they say favours major landowners over small farms.
Earlier this week, dozens of workers of a construction company in central Serbia started a hunger strike, also demanding wages they say have not been paid for a year.
The outgoing government is struggling to keep economic growth forecast at 0.5 percent for 2012 on track and to combat steep unemployment of around 27 percent, while the dinar currency has sunk to record lows this year.
Torn apart by wars in the 1990s, Serbia has since struggled to attract investment to overhaul its ageing infrastructure and create jobs.
The International Monetary Fund earlier this year froze a 1 billion euro ($1.31 billion) standby lending deal with Serbia over its inflated spending plans for 2012. A review of the facility is expected once a new government has taken office and presented a revised 2012 budget.($1 = 0.7622 euros)(Reporting By Aleksandar Vasovic; Editing by Zoran Radosavljevic and Catherine Evans)