Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID) that has been detected widely in surface waters in North America and Europe. The impact of diclofenac on river biofilm communities was investigated at exposures of 10 and 100 μg L−1 of diclofenac or its molar equivalent in carbon and nutrients. Experiments were carried out with river water during spring and summer using rotating annular reactors as model systems. Diclofenac or nutrients at 10 μg L−1 were observed to have no significant effect on algal, bacterial, and cyanobacterial biomass in spring, whereas in the summer the nutrient equivalent reduced algal biomass and diclofenac reduced cyanobacterial biomass relative to control biofilms (p < 0.05). In contrast, at 100 μg L−1 diclofenac or nutrients, the result was increased cyanobacterial and bacterial biomass, respectively, relative to control biofilms in spring. In summer, 100 μg L−1 diclofenac significantly increased bacterial biomass and the nutrient treatment had no significant effect (p < 0.05); both treatments resulted in increased biofilm thickness. The glycoconjugate composition of the exopolysaccharide matrix was influenced differentially by the treatments in both seasons. Biolog assessments of carbon use indicated that 100 μg L−1 diclofenac or nutrients resulted in significant depressions in the use of carbon sources in summer and significant increases in spring. Impacts on protozoan and micrometazoan populations also were assessed. Denaturing gradient gel electrophoresis analyses of community DNA and fluorescent in situ hybridization studies indicated that diclofenac had significant effects on the nature of the bacterial community in comparison with control and nutrient-treated river biofilm communities.