Because the vertical distribution and diversity of blood-sucking flies are poorly known, we determined the diversity, structure, and composition of culicids between vertical vegetation strata. We evaluated the influence of microclimatic variables during different times of the day over a year. We used eight CDC traps baited with CO₂ at a height of 1.5 m and 12-15 m. We conducted rank-abundance curves, similarity analysis (ANOSIM and SIMPER), and multivariate clustering with incidence and abundance data. We used GAM models to analyze the influence of strata (understory vs canopy), humidity, and temperature on insect richness and abundance. During the day, the difference between strata was mainly due to higher abundance of Wyeomyia arthrostigma and Wyeomyia ca. adelpha in the understory. During the night, the differences were mainly due to higher abundance of Culex stigmatosoma, Culex salinarius, and Aedes allotecnon in the canopy, and Wyeomyia arthrostigma in the understory. Seasonality played a role in the similarity between the strata. Diversity during the day was positively related to humidity and temperature, and nocturnal diversity increased with temperature but decreased with higher humidity. The effects of environmental factors on the spatiotemporal distribution of fly species are essential for epidemiological surveillance.