L-asparaginase dominates a great attention due to its biomedical importance as an anti-cancer agent. This study aimed at isolation, screening and characterization of L-asparaginase producing bacteria from Egyptian agricultural soils. Asparaginase production, characterization and optimization by the most potent bacterial isolates were also evaluated. P. carotovorum and S. marcescens are considered as efficient L-asparaginase producing. The L-asparaginase activity of both P. Carotovorum and S. marcescens strains reached 4.835 Uml-1 and 5.221 Uml-1 after optimization with average increase of 7.51% and 23.19% for P. carotovorum and S. marcescens, respectively.