The demonstrations Saturday come just days after Bogota launched peace talks with the country's second-biggest guerrilla group, the National Liberation Army (ELN), setting its sights on a total end to a bloody half-century conflict.
Large crowds ignored heavy rain in some cities as they took to the streets. Many demonstrators were decked out in the yellow, blue and red colors of the Colombian flag.
The protests were called by the Democratic Center party of the conservative former president Alvaro Uribe, who has voiced consistent opposition to negotiations with the Marxist FARC.
The 62 year-old Uribe remains popular and powerful opposition figure. His 2002-2010 presidency was marked by a tough crackdown on the leftist rebels, as well as charges that he favored right-wing paramilitary groups.
In late February Uribe's youngest brother Santiago was arrested on homicide charges linked to a paramilitary hit squad known as the Twelve Apostles. The ex-president claims that Santiago is a "political prisoner."
President Santos, who was Uribe's defense minister, currently suffers from low approval ratings.
"This protest is a general dissent against the government of President Santos. We do not want impunity in the peace processes. We are tired of so many lies and bad economic management, taxes and inflation," Francisco Santos, a former vice president, told AFP.
The biggest turnouts were in the capital Bogota and Medellin, Uribe's hometown, where he led the opposition march.
Police said there was no trouble.
Despite domestic opposition, the peace talks with the Revolutionary Armed Forces of Colombia (FARC) appear to be reaching a successful conclusion after more than three years of negotiations.
Accords bringing in the government and the FARC and ELN would establish peace between the main remaining players in a conflict which over five decades has drawn in right- and left-wing guerrillas, government troops and drug trafficking gangs.
Starting as a peasant uprising in the 1960s, the conflict has killed more than 260,000 people, uprooted 6.6 million people and left a further 45,000 missing.