Fishes were collected in all four streams of the Presque Isle Bay watershed in Erie, PA, to assess the environmental effects of urbanization. These streams were evaluated using an index of biotic integrity (IBI) combined with a multivariate analysis. Four reference streams in adjacent non-urban watersheds were selected for comparison. IBI values and associated impairment status of sites varied from eight sites in three streams that were impaired, to six sites on the four reference streams which were acceptable. The highest diversity sites were the sites farthest downstream in three of the streams, where 6–19 species were collected per site. No fishes were collected in the lowest diversity site and no Pennsylvania species of concern were collected at any sites. Our results indicate that at least half of the stream fish assemblages in the Presque Isle Watershed are impaired, likely due to effects of urbanization.