The Czech Republic currently holds the EU's rotating presidency.
The chamber said milk prices were at their lowest since the Czechs joined the EU in 2004, putting small farms at risk.
Farmers said they would prefer 40 euro cents as a minimum price for a litre of milk, double the 20 cent retail price.
"We are losing terrible amounts of money"
Joseph Rousek, Czech dairy farmer
In addition, they also asked for direct subsidies to dairy farmers or more buyouts from the EU, which would enable the bloc to raise demand and keep up prices.
"We are losing terrible amounts of money. Soon there won't be enough to pay wages. We'll have to slaughter cows, and that is the end," Josef Rousek, a dairy farmer from the town of Bohuslavice nad Metuji, said.
Protesters also dumped milk and manure outside a hotel where Mariann Fischer Boel, the European farm commissioner, and Petr Gandalovic, the Czech farm minister, held a meeting.
Boel met representatives of the farmers and promised further talks in Brussels next month.
The global economic crisis has begun to raise unemployment and public dissatisfaction in the Czech Republic.
|Farmers want to make sure their livelihood and Czech agriculture is protected [Reuters]
Workers from the metallurgic union, the country's largest, have announced another protest for May.
The farmers demanded an immediate increase in EU subsidies, which are currently at a much lower rate for east European producers than for those in the West.
"We want equal conditions by 2010," Jan Veleba, the Czech agrarian chamber chief, told the rally.
"We demand immediate and effective steps to save dairy cows."
The Czech government has pledged $122.9m to help farmers with loan guarantees.