L-asparaginase is an extracellular enzyme that has attracted much attention because, it has highly potential anti-cancerous activity. In the present study, four soil samples each were collected from different areas in and around the karwar regions, Karnataka, India. Out of 16 isolates three bacterial strains (CB1, CB2, MB) isolated from costal and mangrove ecosystem showed maximum L-Asparaginase production than estuarine and terrestrial ecosystems and three actinomycetes (CA, MA, TA) isolated from costal, mangrove and terrestrial ecosystems showed potential L-Asparaginaseproduction than estuarine ecosystems on modified M9 medium. Selected isolates were characterized by biochemical tests.