The enzyme L-asparaginase is commonly used in treatment of acute lymphoblastic leukemia (ALL). Due to its therapeutic importance and growing demands in medical and food industry much of the research have been focused to produce this enzyme with better activity and less disadvantages; for instance microbes have been extensively used to produce L asparaginase at affordable prices, a wide range of bacterial species have been reported that can produce high yields of this enzyme. The present study is an attempt to compare the production of L asparaginases between gram positive and gram negative bacterial species isolated from soil under same fermentation conditions. In this study the productions of L asparaginase from three species of Bacillus namely Bacillus subtilis (LC425423), Bacillus aerophilus (LC425427), Bacillus endophyticus (MG928501) and three strains of Pseudomonas aeruginosa (LC425424, LC425425, LC425426)  was compared. The enzyme activity of L asparaginases produced from Bacillus species were noted as 8.5, 14.3, 7.1 (U/ml). Whereas, the enzyme activity of L asparaginase produced from Pseudomonas aeruginosa strains (LC425424, LC425425, LC425426) was noted as, 8.2, 19.4, 19.1(U/ml) respectively. In this study it was found that Pseudomonas aeruginosa strains yields was more in terms of protein concentration and enzyme activity when compared to Bacillus species. In addition this study also reports the use of modified Brain heart infusion media for screening and isolation of L asparaginase producing bacterial species. It was found that the number of colonies producing L asparaginase in modified Brain heart infusion media was comparatively same when compared to modified M9 media which is extensively used in screening of L asparaginase producers. A total of 159 L asparaginase producers were screened by using two fold dilution technique on modified BHI media while modified M9 media screened 163 colonies producing L asparaginase.