Moscow expelled 20 Czech diplomats on Sunday amid allegations that two Russian spies were behind an explosion that killed two people at a Czech ammunition depot.
The same two spies are also accused of a nerve agent poisoning in Britain in 2018.
Prague had ordered out 18 Russian diplomats on Saturday, prompting the country to retaliate by giving the Czech’s a day to leave.
The Czech Republic said it had informed NATO and EU allies that it suspected Russia of causing the 2014 blast, with EU foreign ministers set to discuss the matter on Monday.
The US State Department commended Prague’s firm response to Russia’s “subversive actions on Czech soil”.
The row is the biggest between Prague and Moscow since the end of decades of Soviet domination of eastern Europe in 1989.
It adds to growing tensions between Russia and the West, raised by its military build-up in Crimea.
Czech Police have identified two men in connection with the blast – Alexander Mishkin and Anatoly Chepigov – who are also accused of involvement in the Salisbury poisonings in 2018.
Czech Prime Minister Andrej Babis said the attack had been aimed at a shipment to a Bulgarian arms trader.
“This was an attack on ammunition that had already been paid for and was being stored for a Bulgarian arms trader,” he said on Czech television.
Russia said Prague’s accusations were absurd as it had previously blamed the blast on the depot’s owners.
It called the expulsions “anti-Russian actions”, accusing Prague of “striving to please the United States”.
British Foreign Secretary Dominic Raab tweeted that the Czechs “have exposed the lengths that the GRU will go to in their attempts to conduct dangerous and malign operations”.