The additional measures affect 11 energy companies, the only firms in Libya's oil and gas sector that had yet to face sanctions, and bring the total number of businesses punished by EU measures to 46.
Thirty-eight people from Gaddafi's inner circle also face a ban on travel to the EU and an asset freeze.
Following a meeting in Luxembourg, EU ministers warned Gaddafi that more punitive measures could be imposed in the future to cut off his access to cash.
"Member states will continue to deprive the regime completely of all funding derived from exports of oil and gas," they said in a statement. "[They] will take additional measures as necessary."
At least 10 EU member states are also participating in NATO-led operations against forces loyal to Gaddafi to enforce a no-fly zone over Libya and an arms embargo, and to conduct air strikes targeting his military infrastructure.
The bloc may also launch a military-backed humanitarian mission with the aim of supplying food, shelter and other essentials to refugee camps on the Tunisian and Egyptian borders sheltering people who have fled violence in Libya.
The mission could also be used to secure transport of supplies directly to Libya, in particular the western city of Misrata, which has been under siege by Gaddafi's forces for weeks.
The EU has yet to complete planning for the mission and is waiting for the United Nations' Office for the Coordination of Humanitarian Affairs to make an official request.
UK wants more powerful strike force
Britain wants NATO to have a more powerful strike force in Libya but whether the alliance steps up attacks will depend on what Muammar Gaddafi's forces do, British Foreign Secretary William Hague said.
Interviewed by Reuters on his way to today's (13 April) international meeting on the Libyan crisis in Qatar, Hague called for intensified sanctions on the Libyan government and for a clear statement that Gaddafi must go.
The Doha meeting also will discuss ways for countries to give money to meet essential needs in opposition-held areas of Libya, Hague said on board a Doha-bound plane late on Tuesday.
"We have sent more ground strike aircraft in order to protect civilians. We do look to other countries to do the same, if necessary, over time," Hague said.
"We would like a continued increase in our [NATO's] capability to protect civilians in Libya," he added.
French Foreign Minister Alain Juppé said on Tuesday that NATO should do more to destroy Gaddafi's heavy weapons. Libyan rebels also have said the alliance is not doing enough.
French Defence Minister Gérard Longuet complained that Paris and London, the nations that launched the first salvos against Gaddafi's regime on 19 March, have been left to bear "the bulk of the effort," AFP reported.
The United States, which initially played a leading role in enforcing the no-fly zone, has withdrawn from ground strikes on Gaddafi's forces while several European allies, such as Italy, put restrictions on the use of their warplanes in Libya.
Britain would like more NATO countries involved to be able to strike targets on the ground, a government source said.
Gaddafi henchman turned negotiator?
Moussa Koussa, a former Libyan foreign minister who fled to Britain last month, also travelled to Doha to meet Libyan rebels on the sidelines of the meeting of the international contact group on Libya.
Scottish police questioned the former spy chief over the 1988 Lockerbie airliner bombing, which killed 270 people, but Britain said on Tuesday he was free to travel, drawing criticism from families of victims.
Hague defended the move, saying "if someone is not under arrest then of course they are free to move around".