Abstract Background In Colombia for several years, the Urabá-Bajo Cauca and Alto Sinú region has registered the highest numbers of malaria cases in the country. Malaria vector incrimination and the characterization of entomological parameters will allow for a better understanding of malaria transmission dynamics and the design of effective vector control strategies for this region. Methods We conducted a longitudinal survey between November 2008 and June 2010 to quantify entomological (abundance and biting activity) and transmission parameters, including infection rate (IR) and entomological inoculation rate (EIR), to incriminate potential anopheline vectors in three localities of a major Colombian malaria endemic region, the Urabá-Bajo Cauca and Alto Sinú: La Capilla, Juan Jose and El Loro. Results A total of 5,316 anopheline mosquitoes corresponding to seven species were collected. Anopheles nuneztovari (69.5%) and Anopheles darlingi (22.2%) were the most abundant species, followed by Anopheles pseudopunctipennis (4.5%), Anopheles albitarsis s.l. (2%), Anopheles triannulatus lineage Northwest (1.8%), Anopheles punctimacula and Anopheles argyritarsis (at < 1%, each). Three species were naturally infected with Plasmodium vivax, An. nuneztovari, An. darlingi (IRs < 1%) and An. triannulatus (IR = 1.5%). Annual EIRs for these species ranged from 3.5 to 4.8 infective bites per year. Conclusions These results indicate that An. nuneztovari and An. darlingi continue to be the most important malaria vectors in this region. Anopheles triannulatus, a species of local importance in other South American countries was found naturally infected with Plasmodium vivax VK247; therefore, further work should be directed to understand if this species has a role in malaria transmission in this region.