The relationships among urbanization, stream hydraulics, and aquatic biology were investigated across a gradient of urbanization in 30 small basins in eastern Wisconsin, USA. Simulation of hydraulic metrics with 1-dimensional unsteady flow models was an effective means for mechanistically coupling the effects of urbanization with stream ecological conditions (i.e., algae, invertebrates, and fish). Urbanization, characterized by household, road, and urban land density, was positively correlated with the lowest shear stress for 2 adjacent transects in a reach for the low-flow summer (p < 0.001) and autumn (p < 0.01) periods. Urbanization also was positively correlated with Reynolds number and % exposed stream bed during months with moderate to low flows. Our study demonstrated the value of temporally and spatially explicit hydraulic models for providing mechanistic insight into the relationships between hydraulic variables and biological responses. For example, the positive correlation between filter-feeding invertebrate richness and minimum 2-transect shear stress observed in our study is consistent with a higher concentration of water-column particulates available for filtration. The strength of correlations between hydraulic and biological metrics is related to the time period (annual, seasonal, or monthly) considered. The hydraulic modeling approach, whether based on hourly or daily flow data, allowed documentation of the effects of a spatially variable response within a reach, and the results suggest that stream response to urbanization varies with hydraulic habitat type.