Human alteration of the stream environment can affect benthic communities in multiple ways. In urban areas, such as southern California, multiple stressors may impair streams simultaneously, as urbanization typically results in degradation of the physical habitat, poor water quality, and high toxicity to benthic organisms. In order to identify which stressors have the greatest impact, we analyzed a dataset of 38 sites from Orange County, California, which included pristine and heavily impaired streams. At each site, benthic invertebrates, water chemistry, and physical habitat data were measured; additionally, water and sediment toxicity was assayed. We evaluated the effects of 17 environmental variables in four categories (i.e., spatiotemporal, physical habitat, water quality, and toxicity) on macroinvertebrate community structure using univariate and multivariate methods. Partial constrained correspondence analysis showed that spatiotemporal factors accounted for the largest portion of the explained variance, reflecting the concentration of minimally impaired sites in the southeastern portion of the county. Of the remaining variable groups, physical habitat explained the largest portion of the variance, followed by water quality and toxicity. Univariate correlations showed that several physical habitat variables had strong relationships with bioassessment scores and other measures of community structure, but few water quality or toxicity variables did. This research will help design future monitoring programs, as well as help set management and restoration priorities by determining which stressors have the greatest impacts on the ecological health of streams.