Brexit Party MEPs turned their backs as the EU's Ode To Joy anthem played at the European Parliament in Strasbourg.
The 29-strong group, including party leader Nigel Farage, Ann Widdecombe and Annunziata Rees-Mogg, performed the stunt as representatives gathered for the first session since the EU elections in May.
In a tweet following the protest, Mr Farage said the move had made the Brexit Party's "presence felt".
His party was not the only one staging a protest today, with Liberal Democrat MEPs wearing bright yellow shirts emblazoned with "stop Brexit" on the front, and "b****cks to Brexit" on the back.
The Brexit Party and Lib Dems - each with opposing views on leaving the EU - came first and second respectively in the EU polls.
Lib Dem MEPs staged their own European Parliament protest
However, the protests did not go down well with some.
Labour MP David Lammy accused the Brexit Party of being "petty".
He wrote on Twitter: "Brexit Party MEPs turning their backs on Europe, as they do their best to isolate the UK from the world. This is petty, small minded little England at its worst. These plonkers do a proud and open nation a disservice. Shame on them."
And Conservative MP Nicholas Soames tweeted: "Truly pathetic oafish childish behaviour by Brexit and Liberal MEPs #growup."
Yesterday, Brexit Party MEP Dr David Bull came under fire after posting a video of himself complaining about the length of his commute to Strasbourg.
Actress Fay Ripley wrote: "Poor guy. He clearly thought Europe was in Guildford."
"Not a Dr of common sense or geography clearly," added Twitter user Steve Box.
Responding to the criticism, Dr Bull said in a statement: "Just to clarify for the unpleasant twitterers. I made that video because the UK voted to leave the EU 3 years ago.
"We shouldn’t have any MEPs. No one should be in Strasbourg. Democracy has been overridden. But now we are there, the @brexitparty_uk MEPs are going to cause a stink."