Pro-Russian rebels in the east have declared independence and have been fighting government troops, leaving more than 400 dead. Tens of thousands have fled their homes.
Targets of the asset freeze and travel ban include two Russian spin doctors, Alexander Borodai, prime minister of the separatist Donetsk People's Republic, and his counterpart in the Luhansk People's Republic, Marat Bashirov.
Both formerly worked for Russian tycoons.
The names were published Saturday in the EU's Official Journal. It brought the total of people affected by the EU's Ukraine-related sanctions to 72, as well as two companies whose EU-based assets have been ordered frozen.
Also on the new list were two men identified as rebel military leaders: Nikolai Kozitsyn, commander of Cossack forces, and Alexei Mosgovy, responsible for military training.
The EU Official Journal said the sanctions, agreed by EU ambassadors on Wednesday, go into effect Saturday.
A Russian diplomat chided Brussels for the move.
Vladimir Chizhov, the Russian envoy to the EU, said in comments carried by Interfax on Saturday that Moscow believes in bringing peace to eastern Ukrainethrough negotiations, and that "blacklisting opponents isn't helping to build a dialogue with them."
Ukraine has refused to negotiate with rebels whom it describes as terrorists, although a Kiev envoy has taken part in a round table with Borodai along with representatives of Russia and the Organization for Security and Cooperation in Europe.
Rebels have rejected Ukraine's call to lay down their arms as a condition for cease-fire negotiations.
In eastern Ukraine, at least four people were killed overnight in Maryinka, a western suburb of Donetsk, when mortar fire hit several apartment blocks near a rebel base, local people said. Four buildings were on fire. An Associated Press journalist saw two bodies.
Local residents, some of whom were fleeing in panic, told the AP that the strikes happened every half hour roughly between 11 p.m. Friday and 4 a.m. Saturday.
It was unclear which side fired the mortars.
Balint Szalko from Donetsk, Ukraine, and Nataliya Vasilyeva from Moscow contributed to this report.