Cyclooxygenase (COX) plays an important role in eicosanoid metabolism. Nonsteroidal anti-inflammatory drugs (NSAIDs) function as COX inhibitors and are frequently detected in the aquatic environment. Here, we measured the in vitro COX-inhibiting activity of the surface water and domestic wastewater in the Tone Canal, Japan. The concentrations of several NSAIDs in the some samples were also determined using gas chromatography-tandem mass spectrometry for confirming the validity of the assay. The target compounds were extracted from the samples using a solid-phase extraction cartridge. A dose-response relationship between the inhibiting activity and sample volume were observed in the wastewater sample. The higher COX-inhibiting activities were observed in the wastewater sample, as compared with the samples of the surface water in the canal. These inhibiting activities reflected the trends of NSAIDs distribution in the canal. However, the inhibiting activities of the water samples could not be entirely explained by the NSAIDs that were selected for instrumental analysis in this study. Other compounds that were not measured by instrumental analysis in this study might contribute to the inhibiting activities. Therefore, the COX-inhibiting assay would be effective for evaluating inclusive ecotoxicity in the aquatic environment.