Barroso said in Croatian to 20,000 people gathered in the Zagreb square hosting the main celebration.
Moments later, the EU anthem, Beethoven's "Ode to Joy" struck up and fireworks lit up the sky above the capital, marking Croatia's entry into the bloc as its 28th member.
Countdown celebrations for the historic moment were held in cities throughout the country of 4.2 million as crowds cheered and waved EU and Croatia's flags in joy.
More than 100 European dignitaries attended the event at the Zagreb main square, where three stages were decked out in the EU's trademark blue for performances by some 700 singers, musicians and dancers.
"Today is a great and joyful day for our homeland...," President Ivo Josipovic told the crowd.
"This the day when we open a new chapter in the thick book of our history."
In a symbolic gesture, on the stroke of midnight Croatia removed the "Customs" sign at a border crossing with Slovenia, the first former Yugoslav republic to have joined the bloc.
At the same time, the "EU" sign went up at the land border with Serbia, another ex-Yugoslav republic, which on Friday got a green light from Brussels to open membership talks by January 2014.
The two events were broadcast live at the Zagreb square, where heads of state from all six ex-Yugoslav republics were among the guests.
Many leaders of EU member states including Britain, France and notably Germany were not there, however.
"I'm so proud, we finally got where we belong," said a 23-year-old student Morana Tokic in Zagreb. Like many in the crowd, she was waving a little flag which on one side had the Croatian flag, and on the other that of the EU.
Biljana Borovec, a 50-year-old clerk, was more cautious. "It yet has to be seen how it will be, it depends on us a lot."
Croatia becomes the first new member to the bloc since Bulgaria and Romania joined in 2007.
But observers say the celebrations were overshadowed by economic worries within the country -- and the EU itself is still struggling with the eurozone debt crisis.
Croatia's tourism-oriented economy has been either in recession or stagnant for the past four years.
EU figures show that the country, where unemployment stands at around 20 percent, is now among the bloc's poorest.