Deforestation, agriculture, farmyard animal husbandry, and urbanization are known to be the main causes of biodiversity loss and habitat fragmentation. The present study evaluated the role of anthropization in modulating Muscidae (Diptera) assemblages in the Humid Chaco ecoregion of Argentina, by testing the biotic homogenization and intermediate disturbance hypotheses. The study focused on natural, rural, and urban habitats in San Lorenzo Department, Chaco Province, where sarcosaprophagous muscid flies were surveyed. A total of 1,343 muscid flies were captured and identified to 7 genera and 24 species and morphospecies. We observed the effect of anthropization on the structuring of the assemblages and the presence of exotic species associated with human activity resulting from biotic homogenization. The highest abundance was recorded in the urban habitat, while the highest species richness and diversity (Shannon-Wienner and Hill's numbers) were found in the rural habitat, supporting the intermediate disturbance hypothesis. Several species are classified as indicators of habitat as well as according to their index of synanthropy. Our results provide valuable information about the use of sarcosaprophagous muscids as indicators of disturbance of natural habitats and about possible health risks related to this family of Calyptratae flies previously unsurveyed in northeastern Argentina. This information could be used in the ecological, agronomy, sanitary, and forensic fields.