We studied a benthic invertebrate assemblage of a stream that passes through pristine, rural, suburban and urban areas of a municipality located in southeastern Brazil to investigate a possible relationship between this assemblage structure and urbanization. The environmental variables and fauna structure were analyzed in a spatial and temporal scale, sampling the four sites in a dry and wet season. We found a clear spatial pattern, with higher similarity between sites from rural and suburban area that presented intermediate environmental characteristics. The pristine site showed in both seasons the lowest values of alkalinity and fecal coliform. On the other hand, the site located in the urban area showed the lowest concentration of dissolved oxygen and higher of suspended solids, ammonia and fecal coliform. The extreme values of these three variables occurred in the wet season, probably related to the high rainfall values. The benthic invertebrate fauna structure followed the same longitudinal and seasonal pattern found for the environmental variables. The site in urban area showed the lowest richness, diversity and evenness, with a dominance of two groups resistant to adverse environmental conditions (Oligochaeta and Orthocladiinae) and absence of more sensitive groups (Coleoptera, Ephemeroptera and Trichoptera). The increase drag of the substrate and associated invertebrates can be responsible for the lower abundance and richness observed in the wet season. The environmental variables that best defined the differentiation between sites (dissolved oxygen, organic suspended solids and fecal coliform) related directly to urbanization effects, like dump effluents and removal of riparian vegetation.