We reanalyzed a dataset consisting of ≈10,700 crepuscular and night-biting female mosquitoes (Culicidae) collected over 12 mo in the canopy and understorey of primary Amazonian rain forest. We investigate whether vertical habitat stratification and rainfall modified major ecological parameters of this mosquito ensemble, combining descriptive and hypothesis-testing statistics with species richness and diversity metrics in the analyses. A total of 31 species was recorded. Contrary to expectations, the host-seeking mosquito fauna was less diverse in the forest canopy than in the understorey. In particular, species diversity and evenness were higher in understorey samples, whereas species richness estimates were similar in both habitats. Only two out of 12 species tested for vertical stratification were clearly acrodendrophilic, and five preferred understorey habitats. The mosquito fauna was more diverse in the rainy than in the dry season. We propose the hypothesis that female mosquito density and host defensive behavior may promote host seeking in nonpreferred habitats by acrodendrophilic mosquito species. These results may be particularly relevant for understanding the dynamics of Plasmodium malariae/brasilianum and arboviral infections in Amazonian forested landscapes.