Background/Aim: Enzymatic anticancer therapies are actively investigated as they can selectively deprive cancer cells of essential nutrients. L-lysine-α-oxidases of different origins have been reported as potential anticancer enzymes with a significant antitumor potency. This study aimed to evaluate the cytotoxic and antitumor activity of L-lysine-α-oxidase obtained from the domestic strain of Trichoderma harzianum Rifai VKPM F-180. Materials and Methods: The melanoma cell line A875 and normal keratinocytes HaCaT were used for cytotoxicity testing. Murine solid tumors sarcoma 45, carcinosarcoma W-256, carcinoma PC-1 and hepatoma 22 were used for experiments with animals. Tumor growth inhibition (TGI) was the primary measure of antitumor efficacy. Results: L-lysine-α-oxidase exhibited selective cytotoxicity toward melanoma cells (IC50=0.09 μg/ml) compared to HaCaT cells (IC50=0.38 μg/ml). In animal models, the enzyme significantly inhibited growth of sarcoma 45, carcinoma PC-1, and hepatoma 22, with TGI ranging from 25% to 41% at 35 U/kg by the 8th or 9th day post-treatment. However, carcinosarcoma W-256, a reactive oxygen species-producing tumor, demonstrated lower sensitivity, particularly at higher enzyme doses. Conclusion: L-lysine-α-oxidase from Trichoderma harzianum Rifai demonstrates promising selective cytotoxicity and antitumor activity, especially in ROS-sensitive tumors.