Among all fungal endophytes isolates derived from different ethno-medical plants, the hyper-yield l-asparaginase and l-glutaminase wild strains Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20 using rice straw under solid-state fermentation (SSF) were selected. The selected strains were used as parents for the intergeneric protoplast fusion program to construct recombinant strain for prompt improvement production of these enzymes in one recombinant strain. Among 21 fusants obtained, the recombinant strain AYA 20-1, with 2.11-fold and 2.58-fold increase in l-asparaginase and l-glutaminase activities more than the parental isolates Trichoderma sp. Gen 9 and Cladosporium sp. Gen 20, respectively, was achieved using rice straw under SSF. Both therapeutic enzymes l-asparaginase and l-glutaminase were purified and characterized from the culture supernatant of the recombinant AYA 20-1 strain with molecular weights of 50.6 and 83.2 kDa, respectively. Both enzymes were not metalloenzymes. Whereas thiol group blocking reagents such as p-chloromercurybenzoate and iodoacetamide totally inhibited l-asparaginase activity, which refer to sulfhydryl groups and cysteine residues involved in its catalytic activity, they have no effect toward l-glutaminase activity. Interestingly, potent anticancer, antioxidant, and antimicrobial activities were detected for both enzymes.