The pharmaceutical concentrations and antimicrobial activity in the Ayase River were investigated. The water sources of the Ayase River are agricultural wastewater and effluent from septic tanks during the irrigation season and effluent from a sewage treatment plant and septic tanks during the non-irrigation season. Acetaminophen, bezafibrate, carbamazepine, clarithromycin, and diphenhydramine levels were in the range of 1.9–495 ng/L. During the irrigation season, pharmaceutical concentrations were higher downstream than upstream due to the inflow of agricultural wastewater in upstream areas. However, during the non-irrigation season, since most of the water sources were effluent from a sewage treatment plant and septic tanks, pharmaceutical concentrations were extremely high from upstream to downstream. The pharmaceutical concentrations during the non-irrigation season were at the same levels as those downstream of the Arakawa River, which was polluted by the effluent from Japan’s largest sewage treatment plant. This extremely high pharmaceutical concentration in the non-irrigated season was found only in the Ayase River but not in the nearby Nakagawa and Furutone rivers, which are both urban water sources. The maximum concentration of clarithromycin in the Ayase River was higher than the predicted no-effect concentration for green algae, indicating that the water from the Ayase River may affect aquatic organisms. Antimicrobial activity analysis in the Ayase River using hypersusceptible Escherichia coli lacking the TolC component of the multidrug efflux system revealed that the river water had antimicrobial activity, suggesting potential risks, such as bactericidal action and development of antibiotic resistance in river water.