This study proposed a battery of endpoints based on in vitro fish cell lines. Two fish cell lines and four toxicity endpoints are considered. The tetracycline (TC), oxytetracycline (OTC), sulfachlorpyridazine (SCP), and septrin(®) (ST) were selected as model antimicrobials and chlorpyrifos as positive control. EROD was induced by septrin(®) (formulation containing sulfamethoxazole and trimethoprim) at concentrations higher than 20mg/L, but inhibited by tetracycline, oxytetracycline and chlorpyrifos. Dose dependent inhibition responses were observed for β-galactosidase (β-Gal) activity in cells exposed to septrin(®), tetracycline or oxytetracycline. The EROD EC50s ranged between 2.29×10(-2)mg/L (chlorpyrifos) and 167.63mg/L (tetracycline). The β-Gal EC50s ranged between 22.1 (septrin(®)) and 84.59mg/L (tetracycline). Data suggest that in vitro testing using a battery of endpoints can be a cost-effective solution for screening the toxicity of antimicrobials on fish. The absence of in vitro effects at concentrations well above those expected/measured in the environment may replace the need for conducting acute lethality tests on fish.