Abstract: Endophytes considered as a potent source of naturally produced bioactive compounds. This study was designed to isolate fungal endophytes that are capable of producing Glutaminase free L-asparaginase enzyme from the selected medicinal plants of Nanded city, Maharashtra, India. Four host medicinal plants traditionally known to have antitumour properties, i.e., Curcuma longa, Murraya koenigii, catharanthus roseus and Withania somnifera were selected for this study. Among the total seventy eight (78) isolated fungal endophytes, only five isolates were shown Glutaminase free L-asparaginase production activity. Fusarium solani isolated from Curcuma longa showedthe highest enzyme production activity level as compared to others by 619.102 IU ml-1 with 8.807 IU mg-1 specific activity,followed by F.oxysporum (514.532 IU ml-1 with 7.401 IU mg-1 specefic activity), Penicillium sp. (307.114 IU ml-1 with 5.033 IU mg-1 specific activity), Aspergillus sp. (216.847 IU ml-1 with 4.920 specific activity ) and Alternaria sp. (149.057 IU ml-1 with 3.035 IU mg-1 specific activity). This study demonstrate the importance of using fungal endophytes from antitumour plant hosts as a good and potent alternative source for L-asparaginase production with novel characteristics at high therapeutic index.