The leaf cutter ant Atta sexdens (L.) (Hymenoptera:Formicidae) causes huge economic losses to agriculture in the Neotropics. In natural environments, parasitic flies of the Phoridae family are natural enemies of leaf cutter ants. Habitat modification is considered one of the main causes of species decline. In this study, we compare the occurrence of parasitic phorids on A. sexdens and the parasitism that they cause on colonies located in agricultural and natural habitats. Phorid flies were collected from trails, nest entrances, and cutting sites when they were hovering over workers of A. sexdens nests from natural vegetation, farmland, and Eucalyptus plantation areas. Simultaneously, workers from A. sexdens nests were collected from these environments, and the parasitism rates of phorids were determined. Ants were parasitized by Apocephalus attophilus, A. vicosae, Eibesfeldtphora bragancai, E. tonhascai, and Myrmosicarius grandicornis. The highest parasitism rate (3.54 ± 0.49%) was registered for nests from the Eucalyptus plantations. The rate of parasitism of nest from natural vegetation was 2.42 ± 0.40% and in the farmland was 1.91 ± 0.39%. The parasitism of each phorid genus varied according to habitat and month. Apocephalus attophilus had the highest parasitism in the three habitats and displayed biological characteristics that give it great potential as a biological control agent for A. sexdens. Eibesfeldtphora spp. had higher parasitism in a natural environment and M. grandicornis in agricultural environments. Apocephalus attophilus and M. grandicornis seemed able to occur in a wide variety of habitats that their hosts occupy, but the rate of parasitism by Eibesfeldtphora spp. was significantly lower in agricultural environments.