Introduction: Elimination of malaria in areas of interrupted transmission warrants careful case assessment to avoid the reintroduction of this disease. Occasional malaria cases are reported among visitors of the Atlantic Forest area of Brazil, while data on residents of this area are scarce. Methods: A sectional study was carried out to examine 324 individuals living in a municipality where autochthonous cases were detected. Results: Asymptomatic Plasmodium infections were detected in 2.8% of the individuals by polymerase chain reaction (PCR), with one case of P. falciparum (0.3%), two cases of P. vivax (0.6%), and six cases of P. malariae (1.9%). The thick blood smears were negative in all individuals. Serological tests performed in 314 subjects were reactive in 11.1%, with 3.5% for P. falciparum and 7.7% for P. vivax. A subsample of 42 reactive individuals for any Plasmodium species showed P. malariae in 30.9% of specimens. Individuals who entered the Atlantic Forest region were 2.7 times more likely to exhibit reactive serology for P. vivax compared with individuals who did not enter this region (p<0.05). Children <15 years had a higher chance of reactive serology for P. falciparum and P. vivax than individuals ≥15 years of age (p<0.05). Individuals living in the Paraiso district had a higher chance of reactive serology for P. vivax compared to other districts (p<0.05). No associations were found between sex, past exposure to malaria, or serological response to antibodies of any Plasmodium species. Conclusions: The implications of these results for the elimination of malaria were discussed. All the procedures followed the ethical standards of the Ethics Committee of Research involving Human Subjects of the Research Institute Evandro Chagas-INI/Fiocruz, Brazil and in accordance with the Helsinki Declaration (Protocol
0229.0.000.009/10 approved).