Background <p>Recent statistical analyses indicate a rapid increase in the incidence of breast and colon cancer in Egypt. Although invasive techniques have been widely employed for early detection, diagnosis, and intervention of those cancers, they are associated with inherent risks and limitations, which often result in various complications. Therefore, noninvasive screening methods are inevitable due to their accessibility, cost-effectiveness, and high patient compliance rates. The enzyme L-asparaginase catalyzes the conversion of L-asparagine to L-aspartic acid: key metabolite for tumor cell division, thereby demonstrating anticancer potential. However, the prolonged use of bacterial L-asparaginase may cause allergic reactions and side effects such as diabetes, leukopenia, and co-agglutination disorders. Exploring the anticancer properties of L-asparaginase from different species such as yeast and fungi has been proposed to mitigate these adverse effects.</p> Objectives <p>This study aimed at extracting and optimizing the expression of L-asparaginase from the eukaryotic <i>Cladosporium</i> species, as to assess its anticancer potential against breast and colon cancer cell lines.</p> Method <p><i>Cladosporium</i> species were identified morphologically and then cultured on modified&#xa0;Czapek-Dox Agar (mCDA)&#xa0;medium supplemented with L-asparagine to induce L-asparaginase production. Submerged fermentation was employed to optimize enzyme production. The enzyme activity was quantified using the Nesslerization method, and its cytotoxicity against colon and breast cancer cell lines was assessed using the (MTT) assay.</p> Results <p>Among the <i>Cladosporium</i> isolates, 18.4% exhibited positive plate assay test, with enzyme activities ranging from 255 to 428 U/mL. Immunoblotting using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed single protein band of approximately 37&#xa0;kDa, consistent with L-asparaginase activity. Cytotoxicity assay of purified L-asparaginase showed significant antiproliferative effects against breast cancer cell lines MCF-7 and MDA-MB-231, with IC₅₀ values of 36.26 and 45.7&#xa0;µg/mL, respectively.</p> Conclusion <p>Certain eukaryotic <i>Cladosporium</i> strains are potential sources for the anticancer L-asparaginase production.</p> Graphical Abstract <p>Microbial L-asparaginase is one of the most important industrial enzymes of interest accounting for about 40 % of the total worldwide enzyme sales, this enzyme has got much significance in&#xa0;the medical field for the treatment of leukemia especially acute lymphoblastic leukemia (ALL).</p> <p></p>