The aim of the present work was to study the in-vitro cytotoxic effects of different concentrations of aflatoxin B1 (AFB1) on broiler lymphocytes. Lymphocyte-rich mononuclear cells were separated by Ficoll-Histopaque density and cultured in 96-wellplates containing the evaluated AFB1 concentrations in 5% CO2 atmosphere at 39°C. Thereafter, MTT, PicoGreen, and reactive oxygen species assays were performed. Cell viability decreased in the presence of 10 µg/mL AFB1 at 48 h (p < 0.05) and of 10 and 20 µg/mL AFB1 at 72 h (p < 0.01 and p < 0.001, respectively) when compared to the control (0 µg/mL). However, a dose-dependent increase in the cell-free DNA at 24 h was observed at 1, 10 and 20 µg/mL (p < 0.001). ROS formation significantly increased at 24 h at all concentrations (p < 0.001). The in-vitro results demonstrate that AFB1 is cytotoxic and causes biomolecular oxidative damage in broiler lymphocytes.