And he urged his MEPs to reject the bid by Germany’s new eurosceptic party, Alternative für Deutschland (AfD), to join the ECR group ahead of Thursday’s vote.
But Britain’s Conservatives lost six seats in the Parliament following May’s European elections, meaning they can be out voted in the group.
Some of them, however, appeared to have ignored his pleas and voted, along with their colleagues from other countries, to admit the AfD.
The AfD said it was voted in with a "clear majority", while Conservative MEP for Eastern England David Campbell Bannerman described the vote as "close" on Twitter. The vote passed by narrow majority of 26 against and 29 for.
Breaking News!: ECR Group currently 3rd largest group in Euro Parliament with 63 members, more than ALDE. AfD joins ECR in close vote.
— D Campbell Bannerman (@DCBMEP) June 12, 2014
Welcome to the @AfD_Bund party which has joined the ECR Group this morning.
— ECR Group (@ecrgroup) June 12, 2014
Professor Michael Wohlgemuth, from think-tank Open Europe Berlin, said the vote was embarrassing for Cameron.
“It is certainly bad for Cameron if he wants to show he is close to Merkel," he told The Local. "He now has one of Merkel’s fiercest enemies as an ally.”
The alliance with the AfD, who want to scrap the euro, will upset Merkel, whose support Cameron needs to renegotiate Britain’s relationship with the EU and see off his eurosceptic rivals UKIP.
He is also seeking the German chancellor's support to stop Jean-Claude Juncker becoming the next president of the European Commission.
A Conservative Party spokesman said: "We are very disappointed that AfD have been admitted into the ECR against our wishes. We note that the vote was a close one. We will work with the AfD in the European parliament but the CDU/CSU remains our only sister party in Germany."
The AfD, which only formed in February last year, took seven percent of the vote in Germany in May's EU elections and gained support from former voters of Merkel's conservative bloc.