The relationship between deforestation and malaria is a spatiotemporal process of variation in Plasmodium incidence in human-dominated Amazonian rural environments. The present study aimed to assess the underlying mechanisms of malarial exposure risk at a fine scale in 5-km2 sites across the Brazilian Amazon, using field-collected data with a longitudinal spatiotemporally structured approach. Anopheline mosquitoes were sampled from 80 sites to investigate the Plasmodium infection rate in mosquito communities and to estimate the malaria exposure risk in rural landscapes. The remaining amount of forest cover (accumulated deforestation) and the deforestation timeline were estimated in each site to represent the main parameters of both the frontier malaria hypothesis and an alternate scenario, the deforestation-malaria hypothesis, proposed herein. The maximum frequency of pathogenic sites occurred at the intermediate forest cover level (50% of accumulated deforestation) at two temporal deforestation peaks, e.g., 10 and 35 years after the beginning of the organization of a settlement. The incidence density of infected anophelines in sites where the original forest cover decreased by more than 50% in the first 25 years of settlement development was at least twice as high as the incidence density calculated for the other sites studied (adjusted incidence density ratio = 2.25; 95% CI, 1.38-3.68; p = 0.001). The results of this study support the frontier malaria as a unifying hypothesis for explaining malaria emergence and for designing specific control interventions in the Brazilian Amazon.