To provide information for public health policy on mosquito nets in the Amazon region of Colombia, we conducted landing catches to estimate Anopheles species composition and biting activity. Two hundred twenty person-nights of catches were done in seven locations over a period of 14 mo. A total of 1,780 Anopheles mosquitoes were caught (8.1 per person-night). Among the nine species found, An. oswaldoi Peryassú was the most common (776 mosquitoes, 44%), followed by An. darlingi Root s.l. (498, 28%). An. oswaldoi was the most common species collected outdoors, where its biting rate dropped steadily from a peak of >15 bites/person-night at the start of the night (1800–1900 hours) to ≈2 bites/person-night before dawn. An. darlingi was the most common species collected indoors, with a biting rate of ≈3–4 bites/person-night until about midnight, when the rate dropped below 1 bite/person-night, before showing a secondary peak before dawn. Sixty-four mosquito nets were analyzed by the technique of high-performance liquid chromatography (HPLC) for levels of deltamethrin (DM). All but two (62) of these were reported by their owners to have been impregnated with insecticide, and 53 were found by HPLC to have deltamethrin. However, one half (32) of the nets had concentrations <4 mg/m2 and therefore were likely to have been inadequately protective. An inverse association was found between the reported time between washes and deltamethrin concentration. These findings show a need for additional protection from mosquitoes when not inside nets, as well as for more effective impregnation, possibly through wash-resistant insecticide formulation.