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Benzothiazoles are high production volume chemicals widely used in many industrial and household applications. However, information on their occurrence in aquatic organisms is very limited, although a high level of bioaccumulation is expected. In this study and for the first time, a method was developed involving subcritical water extraction followed by solid-phase microextraction coupled to gas chromatography-ion trap-tandem mass spectrometry for the determination of five benzothiazoles in seafood. The repeatability and reproducibility of the method were under 21% (%RSD, n = 5, 100 ng g−1 (dw)), while method detection limits and method quantification limits were between 0.5 and 10 ng g−1 (dw) and 1 and 50 ng g−1 (dw), respectively. Ten widely consumed fish and shellfish species from the county of Tarragona (Catalonia, Spain) were selected in order to estimate dietary exposure and to assess the human health risks. The most frequently determined compounds were benzothiazole and 2-(methylthio)-benzothiazole, with squid being the species which showed the highest level of benzothiazole (82 ng g−1 (dw)). In terms of human exposure, the current concentrations of benzothiazoles found in fish and shellfish could not be compared to threshold values because of the lack of toxicological data.