In the malaria endemic municipality of Miraflores in southeastern Amazonian Colombia, several aspects of the biology of local Anopheles species were investigated to supplement the limited entomological surveillance information available and to provide baseline data for malaria prevention and vector control. Anopheles darlingi Root, 1926 was the most abundant species (95.6%), followed by Anopheles braziliensis (Chagas) (3.6%) and Anopheles oswaldoi s.l. (Peryassu) (0.7%). During the dry season, exophagic activity was prevalent only between 1800–2100 hours; after this (2100-0600 hours) only endophagy was encountered. In contrast, during the rainy season, both endophagy and exophagy occurred throughout the collection period. The human biting rate for An. darlingi was 8.6. This species was positive for Plasmodium vivax VK210 with a sporozoite rate = 0.13 (1/788). Breeding sites corresponded to stream (n = 7), flooded excavations (n = 4), flooded forest (n = 1), wetlands (n = 2), and an abandoned water reservoir (n = 1). An. darlingi predominated in these sites in both seasons. Based on these data, An. darlingi is the main local malaria vector, and we recommend that local prevention and control efforts focus on strengthening entomological surveillance to determine potential changes of species biting behavior and time to reduce human-vector interactions.