Introduction: L-asparaginase is a therapeutic enzyme that is used in combination therapy for mainly Acute Lymphocytic Leukemia (ALL) and other cancers. It hydrolyzes L-Asparagine required by neoplastic cells into L-aspartate and ammonia which leads to the death of tumor cells. But commercially available L-asparaginases show fatal side effects because of its prokaryotic source of origin, and associated glutaminase and urease co-activities. Aims: Screening of marine fungi for L- asparaginase activity and study of its potential as an anti-cancer agent. Methodology: This study was designed to isolate fungi from marine water and soil samples collected from regions of Maharashtra__ampersandsignnbsp;and Goa, that are capable of producing L-asparaginase enzymes that are free of urease and glutaminase activity, and further study the potential of anticancer activity of these eucaryotic enzymes using Chorioallantoic membrane (CAM) assay. Result: Among all twelve marine fungi isolated, RF2 (identified as Aspergillusaculeatus (Genbank accession no.ON023823)) from Mandvi beach, Ratnagiri MH demonstrated high levels of L-asparaginase activity within 48-96 hrs, while lacking glutaminase and urease activity. Conclusion: The Chorioallantoic membrane (CAM) assay revealed that this fungal isolate could be a promising source of eucaryotic L-asparaginase to treat cancer with lower side effects.