The increasing worldwide production of bisphenols has been associated to several human diseases, such as chronic respiratory and kidney diseases, diabetes, breast cancer, prostate cancer, behavioral troubles and reproductive disorders in both sexes. The aim of the present in vitro study was to evaluate the potential impact bisphenols A, B, S and F on the cell viability and testosterone release in TM3 Leydig cell line. Mice Leydig cells were cultured in the presence of different concentrations of bisphenols (0.04-50 µg.ml-1) during 24 h exposure. Quantification of the cell viability was assessed using the metabolic activity assay, while the level of testosterone in cell culture media was determined by enzyme-linked immunosorbent assay. Within the panel of substances under investigations, the higher experimental concentrations (10; 25 and 50 µg.ml-1) significantly (P<0.001) decreased Leydig cells viability, while the same doses of BPA and BPB also reduced testosterone production significantly (P<0.001). Taken together, the results of our study reported herein is a consistent whit the conclusion that higher experimental doses of bisphenols have a cytotoxic effect and could have a dose-dependent impact on testosterone production.