Abstract While the antimicrobial ingredient triclosan has been widely monitored in the environment, much less is known about the occurrence and toxicity of its major transformation product, methyl triclosan. An improved method was developed and validated to effectively extract and quantify both contaminants in fish tissue and used to characterize concentrations in small prey fish in areas of San Francisco Bay where exposure to triclosan via municipal wastewater discharges was expected to be highest. Concentrations of triclosan (0.44–57 ng/g wet wt, median 1.9 ng/g wet wt) and methyl triclosan (1.1–200 ng/g wet wt, median 36 ng/g wet wt) in fish tissue decreased linearly with concentrations of nitrate in site water, used as indicators of wastewater influence. The total concentrations of triclosan and methyl triclosan measured in prey fish were below available toxicity thresholds for triclosan, but there are few ecotoxicological studies to evaluate impacts of methyl triclosan. Methyl triclosan represented up to 96% of the total concentrations observed. These results emphasize the importance of monitoring contaminant transformation products, which can be present at higher levels than the parent compound. Environ Toxicol Chem 2023;42:620–627. © 2023 SETAC