L-glutaminase is an enzymatic catalyst that hydrolyzes glutamine, converting it into L-glutamic acid and ammonia. It has several biotechnological uses in the medicinal and food sectors. Moreover, its role in enzyme therapy for cancer treatment, particularly in acute lymphocytic leukemia, has been widely acknowledged. This study screened marine sediment-derived actinobacterial isolates for L-glutaminase. The most promising L-glutaminase producing strain was selected and identified through 16S rRNA gene sequence analysis as Streptomyces griseorubens NAHE (OR462786). The maximum L-glutaminase activity (20.777U/ mL) was detected in the growth medium containing sucrose and glutamine as the carbon and nitrogen sources, respectively. The Plackett-Burman experimental design was used to optimize and identify the factors that have the greatest impact on L-glutaminase production. This design revealed that the optimized medium increased L-glutaminase activity by 1.47-fold, higher than that recorded in the case of the basal cultural conditions. The current study also showed that L-glutaminase produced by adsorbed marine S. griseorubens NAHE enhanced enzyme activity by 3.31-fold compared to conventional free cells. Furthermore, the produced enzyme exhibited a promising antimicrobial activity against Staphylococcus aureus, Fusarium oxysporium, Candida albicans, and Aspergillus flavus, which indicates its suitability for numerous therapeutic applications.