Manuel Rosales, the governor of the oil-rich Zulia state, was chosen on Wednesday to challenge Chavez in December’s presidential election.
Eight other hopefuls stepped aside to unite behind Rosales.
Rosales said: "Today we open the path of hope, of national reconciliation, of the fight for a better Venezuela."
The move to back Rosales is likely to lead to the cancellation of proposed opposition primaries that had been scheduled for mid-August.
Venezuela's opposition has been severely fractured since defeat to Chavez in 2004.
Opposition parties pulled out of the December 2005 parliamentary elections citing concerns about electoral fraud, handing Chavez 100 percent control over the nation's legislature.
Accion Democratica, once Venezuela's strongest political party which used to count Rosales as a member, has called for abstention in the upcoming elections.
The unity accord did not cover all opposition candidates. Benjamin Rausseo, a stand-up comedian who recently announced his intention to run for president, did not join the agreement to back Rosales.
Other opposition splinter candidates are also expected to run.
Chavez, a former soldier who has promised to end poverty in the country which is the world's fifth largest oil exporter, is expected to win re-election after cultivating widespread support by spending billions of dollars in oil revenue on social programmes.