The objective of this study was to extract and characterize L-asparaginase from a thermophilic fungus, Aspergillus fumigatus which was isolated from different soil samples, and explore its antimicrobial activity. Six strains of fungi were isolated from soil samples at Mansoura University, identified, and screened for their ability to produce L-asparaginase using a modified Czapek-Dox medium with L-asparagine. Among the isolated fungi, Aspergillus fumigatus exhibited the highest L-asparaginase activity, identified through screening on Czapek's agar media. The organism was cultivated under submerged fermentation conditions, yielding L-asparaginase at its maximum activity at 40 °C and pH 8.0 after 30 minutes. The findings suggest that Aspergillus fumigatus from the soil samples has significant L-asparaginase activity and could be further explored for commercial L-asparagine production, given the enzyme's effective use as an antibacterial agent.