Bioactive components derived from medicinal herbs have recently acquired popularity due to their efficacy in treating various ailments, including cancer and infectious diseases. In this study, the anticancer enzyme, L-methioninase isolated from medicinal plants endophytic fungi, then evaluated its promising therapeutic agents against different types of human cancers. L methionine was purified using column chromatography with the stationary phase of Sephadex G-200 with 6.6-fold purification, which increased the specific activity of 71.3 U/mg of protein with a recovery rate of 48.2 %. On the SDS-PAGE chromatogram, the apparent molecular mass of the isolated enzyme was 48 kDa, and its highest activity was observed at pH 8 and 35 °C. The enzyme was catalytically stable within the pH range of 6.0–9.0 and below 40 °C. This study demonstrates that isolated L-methioninase is particularly efficient against tumour cell lines in vitro. The crude and purified L-methioninase inhibited 60 and 80 % of the growth of the breast cancer cell line (MCF-7), respectively, with an estimated IC50 = 12.6 μg/ml (crude) and IC50 = 5.0 μg/ml for purified L-methioninase from isolate 8 with accession no MZ675362. Because of this, pure L-methioninase has better catalytic characteristics and significant thermal stability, which could be used as a cancer-fighting substance than the enzyme purified from other sources.