Proteases are highly required where proteins need some sort of alteration either structurally or functionally. They are widely used in household laundry, paper and leather industries as well as in several pharmaceutical compounds. Thus, the present study was confined to screen more than fifty isolates of microbes from the rhizospheres of fruit orchard for proteolytic activity on skimmed milk agar medium. Isolates SK701 and SK709 were found producing halo regions around the streaked mycelia. However, only isolate SK709 displayed maximum 64.6 U/ ml protease activities in starch casein broth after 96 hours of culture at optimum conditions of 35°C and pH 7.6. It was identified as a gramnegative bacterium on the basis of staining, morphological, cultural, biochemical and physiological characteristics. The cytotoxic activity of cell free extracts of isolate SK709 against EAC, SiHa & Hep G 2 cell lines in terms of IC 50 was found to be 15.1, 26.3 and 35.5µg/ml, respectively. The 16S rRNA gene sequencing, molecular characterization and the BLAST operation as per NCBI directive to construct phylogenetic tree confirmed the putative protease producing isolate SK709 to be the species of Stenotrophomonas. The difference of 2% in the gene sequence with the closest member, Stenotrophomonas sp. MCYF1(KC734883.1) which amounted to 21 out of 1022 nucleotides, made the isolate SK709 qualified for obtaining a separate NCBI accession number KF484911..