The current study describes the eco-epidemiological aspects of arbovirus diseases in the municipalities (counties) of Novo Progresso and Trairão, Para State, Brazil, in the area affected by highway BR-163. Hemagglutination inhibition (HI) antibodies to different arboviruses were detected, with monotypic reactions to MAYV and OROV, two important arboviruses associated with epidemics in the Amazon. IgM antibodies to OROV and MAYV were found in human sera, suggesting recent infections by these viruses. Two DENV-3 strains were isolated from febrile patients in Novo Progresso and identified as genotype III strains. In general, the data suggest that the area displays ideal conditions for maintenance and circulation of arboviruses, plus a population with low immunization levels. Dynamic surveillance of local immigrants and wild animals is thus important, focusing on antibody prevalence and isolation of arboviruses, thereby allowing effective control of infections by these viral agents in the resident population along highway BR-163 in Pará State.