The assassination of left-wing politician Chokri Belaid in July - the second such killing in five months - has plunged the country into crisis with the opposition accusing the government of failing to maintain security and kickstart the economy.
A coalition of opposition parties known as the National Salvation Front is calling for a politically independent government to run the country and organise new elections.
"We tried you, you failed, now leave," chanted protesters in the first demonstration of what is being called by the opposition the "week of departure" for the government.
Protesters began by singing the national anthem led by opposition assembly members who have stopped participating in the elected body in protest at the government's performance.
Many were surrounded by bodyguards in response to death threats.
Police who monitored the demonstration checked people's bags, but there were none of the clashes or tear gas that have marred past protests.
Tunisia's UGTT trade union has been mediating between the Islamists and the opposition.
The ruling Ennahda party said on Thursday it accepted in principle a proposal to form a technocratic government, but only after further negotiations.
But the secular opposition has condemned the Islamists' move as merely a stalling tactic and maintains that dissolving the government is a prerequisite for further talks.
"The opposition is determined to say No to any negotiations before the current government is dissolved," said Karima Souid, a member of the left-wing Al-Massar party.
"We call for a government of public salvation to manage the country's affairs and carry out free and fair elections, without fear."
Tunisia was the birthplace for the Arab Spring uprisings when it overthrew long-ruling president Zine el-Abidine Ben Ali in January 2011.
The rocky transition to democracy has included political assassinations, terrorist attacks and unrest over a faltering economy which Ennadha has failed to transform.