L-Asparaginase is an enzyme (EC3.5.1.1) used as chemotherapeutic agent for the treatment of Acute Lymphoblastic Leukemia (ALL). The commercial L-Asparaginase now available is of bacterial origin and since it shows many side effects, search for alternative sources of this enzyme is highly necessary, owing to its high therapeutic significance. In the present study an attempt has been made to isolate bacterial endophytes, with L-Asparaginase potentiality, from medicinal plants. From 16 different medicinal plants 127 bacterial endophytes have been isolated, among which 51 endophytes were capable of producing L-Asparaginase enzyme. Two isolates that showed excellent enzyme activity as 1.436 U/ml and 1.827 U/ml were identified, based on biochemical tests and 16s rRNA gene sequencing, as Acinetobacter baumannii and Bacillus subtilis which were isolated from the medicinal plants, Annona muricata and Averrhoa carambola respectively. The phylogenetic tree for the two isolates were constructed based on the 16s rRNA blast results and the 16s rRNA gene sequences were deposited in gene bank and accession numbers were received.