L-ASPARAGINASE enzyme is important medically as an anticancer agent and in the food industry. The enzyme acts via degradation of l-asparagine and mitigation of acrylamide. This work screened 31 fungal isolates recovered from rhizosphere soil for l-asparaginase production using the plate dilution medthod. Twenty-four isolates (77.4%) were l-asparaginase producers. Aspergillus niger and A. quadrilineatus were the highest producers with enzyme activities were 9.808±0.18930 and 7.348±0.12328U/mL, respectively. Optimum conditions for enzyme production were 30°C for 72h, with pH 6 at 160rpm, and 0.1% of KH2PO4 in presence of 2% glucose and 1.5% sucrose as carbon source and 1% L-asparagine by A. niger and A. quadrilineatus, respectively. Ammonium sulfate precipitation, Sphedax G-200, and SDS-PAGE were performed for L- asparaginase purification and molecular weight determination. Enzyme from A. niger displayed a MW of 50.36kDa and a specific activity of 50.4U/mg. The MW of A. quadrilineatus enzyme was 27.8kDa with a specific activity 37.4U/mg. Purified l-asparaginase significantly inhibited the proliferation of HCT-116, HePG-2, and MCF-7cells with IC50 concentrations of 28.9, 36.1, and 82.6μg/mL, respectively. The enzyme did not exhibit antibacterial activity. Enhancement of l-asparaginase production using agro-industrial wastes produces a maximum of 23.548 ± 0.00000U/mL when A. niger is cultivated on a mixture of onion and pomegranate peel powders (50%: 50% w/w) and cultivation of A. quadrilineatus on pomegranate peel alone.