This study was conducted to determine Anopheles species composition and their natural infectivity by human Plasmodium in 2 localities with the highest malaria transmission in San Jose del Guaviare, Guaviare, Colombia. A total of 1,009 Anopheles mosquitoes were collected using human landing catches during 8 months in 2010. Anopheles darlingi was the most abundant (83.2%) followed by An. albitarsis s.l. (8.6%), Anopheles braziliensis (3.8%), An. oswaldoi s.l. (1%), and An. rangeli (0.3%). Anopheles darlingi showed the highest human biting rate, and it was found naturally infected with Plasmodium vivax VK210 (0.119%) using enzyme-linked immunosorbent assays. All species were collected biting both indoors and outdoors. Anopheles darlingi showed biting activity overnight with an indoor peak between 1200–0100 h. Therefore, we recommend that malaria prevention strategies focus on 1) insecticide-treated nets to reduce human–vector contact when people are most exposed and unprotected; 2) accurate diagnoses; 3) adequate treatment for patients; 4) more timely epidemiological notification; and 5) improved entomological surveillance.