Understanding the changes in biodiversity correlated with urbanization is essential for monitoring the complex effects of human activity on native ecosystems. We hypothesized that the Drosophila community native to temperate woodland forests would change along a gradient of urbanization, and could therefore serve as a model system in studies on urbanization. We used an urbanization gradient we had previously characterized in Southwest Ohio. Community composition gradually changed along the gradient, although community diversity did not. Abundance varied significantly among sites, with one species, Drosophila melanogaster, increasing in abundance from the least to the most urbanized sites. We used 28 parameters from three sets of environmental data—land cover, vegetation, and temperature and humidity—to build a model with Canonical Correspondence Analysis and characterize the species-environment relationship. The most predictive variables explaining the distribution of the Drosophila community were maximum temperature, maximum saturation deficit, percent lawn cover, average diameter at breast height (dbh) of shrubs and trees, and number of tree species. We conclude that the presence of individual, easily identifiable Drosophila species may serve as robust indicators of the habitat degradation brought about by urbanization, and as ideal models for exploring animal response to urbanization.