Experimental studies evaluating the simultaneous effects of consumers, nutrients, and other biotic/abiotic factors on intact, natural food webs are rare, particularly among ecosystems of varying trophic conditions. We conducted a series of in situ studies that used nutrient-diffusing substrata with nitrogen (N) and phosphorus (P) concentrations in a full factorial design in three temperate, limestone streams in Pennsylvania across a trophic gradient (mesotrophic, eutrophic, and hypereutrophic streams). We assessed differences in algal and macroinvertebrate biomass, taxonomic composition, and functional groups relative to amended nutrients across the trophic gradient; as such, these results facilitated predictions about regulators of food web structure. All factors varied significantly among the streams (e.g., algal biomass P = 0.005, macroinvertebrate biomass P < 0.001, algal diversity P = 0.006, macroinvertebrate diversity P < 0.001, algal group P < 0.001, macroinvertebrate guilds P < 0.001); the streams, however, did not exhibit simple responses to nutrient amendment. Algal and macroinvertebrate biomass and diversity responded greatest in the mesotrophic stream while grazing seemed to be a strong factor preventing algal nutrient response in the eutrophic and hypereutrophic streams. Brillouin's Evenness Index was most influenced by nutrient amendment (nutrient effect on algae and macroinvertebrates P = 0.021). As such, we concluded that biomass and diversity were mediated by complexity within intermediate trophic levels.