Marine fungi are important sources of new metabolites including certain enzymes of medical interest due to their enormous capacity to adapt themselves to extreme environments. Living in a highly competitive ecological niche, they produce certain unusual chemical moieties. Marine biological resources are green, abundant, renewable and aid in economic development. The present study investigates the production of L-Glutaminase which is of therapeutic and industrial importance, from marine fungi of coastal Karnataka. Primary screening on agar plates and submerged fermentation in broth was employed for enzyme production. Both marine yeasts (Pichia sp) and filamentous fungal strains (Aspergillus, Penicillium) were found to be efficient producers of L-Glutaminase. Of the 42 isolates, five potential strains were selected through primary screening and Thin Layer Chromatography was performed to confirm the production. Filamentous fungi were identified through morphological and molecular methods as Penicillium and Aspergillus strains with 99-100% similarity. A. foveolatus (MT667385)and A. nidulans (MT667422) were potential producers (1.58U/ml and 1.41IU/ml). The yeast identified was Pichia kudriavzevii (MT667428), which was a moderate producer of Glutaminase and first marine yeast reported for this enzyme production. Neosartorya quadricincta (MT667427) and P. citrinum (MT667426) are also moderate producers. After screening the marine fungi, the isolated strains’ potential to produce L-Glutaminase was confirmed using SDS PAGE, FTIR and Mass analysis. This study emphasizes the necessity of marine fungal culturing and the scope of use of these fungi for further commercial production of L-Glutaminase which would uplift marine economy.