Members in the industry and environment committees voted overwhelmingly against Bratušek - by 112 to 13 votes, after what was widely considered as the worst hearing among Juncker's 27 commissioners.
Bratusek, a former prime minister is under investigation at home for conflict of interest as she appointed herself as commissioner nominee while still in office.
She was slated to become one of Jean-Claude Juncker's seven senior commissioners, in charge of the nascent 'energy union'.
Juncker may try and play hardball with the European Parliament, who can only vote down the entire commission.
But MEPs who were part of the discussions with Juncker doubt he will continue to back her after the negative vote Wednesday evening, according to EUobserver.
Lithuanian MEPs have also expressed reservations about Bratušek's candidacy.
"Even before the official hearings, she was not considered the strongest candidate. We have to acknowledge that during the hearings Ms. Bratušek, unfortunately, did not present herself from the best side, including her language skills, as she has not got a very good command of English. Her answers also did not convince the committee members," Petras Auštrevičius of the Group of the Alliance of Liberals and Democrats for Europe told BNS on Wednesday.
Zigmantas Balčytis of the Group of the Progressive Alliance of Socialists and Democrats in the European Parliament also commented to BNS that Slovenia should look for a new candidate as Bratušek, in his opinion, was not competent enough to hold such a key position.
On 27 May Lithuanian government has allowed renewing more flights to Germany and the Netherlands .