This study was conducted to quantitatively evaluate the antioxidant, antihemolytic and nephroprotective effects of Diospyros lotus seeds extract in experimental in vitro and in vivo models. Antioxidant potential of Diospvyos lotus seeds extract was examined by employing seven in vito models i.e., DPPH, nitric oxide and hydrogen peroxide radicals scavenging activity, iron ion chelating, reducing power and lipid peroxidation through linoleic acid. Antihemolytic activity of extract was examined against hydrogen peroxide-induced erythrocytes hemolysis. Also, nephroprotective effect of extract against gentamicin (GM)-induced renal injury was evaluated. Renal injury was achieved by injecting 100 mg/kg, intraperitoneally (i.p.) of GM in normal saline. Extracts were administrated i.p. in doses 200 and 400 mg/kg. Blood samples were examined for serum creatinine and blood urea nitrogen after 10 consecutive days of treatment. Results show that extract showed different level of antioxidant and antihemolytic activity in the studied models. Also, results show that GM-induced nephrotoxic animal model was successfully constructed. Extract attenuated the gentamicin-induced increase in level of serum creatinine and blood urea nitrogen. The present study shows that the extract offered significant biological action compared with standard compound.