We integrated the results of field surveys with a mesocosm experiment to quantify the ecological impacts of a gasoline and diesel spill on a third-order stream in western Colorado (United States). The spill caused a massive fish-kill of brown trout ( Salmo trutta) and mottled sculpin ( Cottus bairdii), which extended several kilometers downstream. Despite significant decreases in petroleum hydrocarbon concentrations, subsequent surveys indicated little recovery of fish populations 4 years after the spill. Benthic macroinvertebrate communities were also affected, yet some metrics commonly used to assess stream ecological integrity (e.g., total abundance and species richness) showed no difference between impacted and reference sites. The failure of some groups to recover 2.5 years after the spill was likely a result of their comparatively slow reproduction and recolonization rates. To support our hypothesis that effects observed in the field resulted from petroleum exposure, we conducted a mesocosm experiment in which benthic macroinvertebrate communities were exposed to a simulated diesel spill. We observed significant decreases in the abundance of most macroinvertebrate groups at the lowest exposure concentration (75 mg/L diesel) and a strong concentration-dependent drift response across all groups. Our study suggests that relatively small petroleum spills can significantly affect stream communities, and these effects may persist several years after sediment concentrations return to background levels.