The objective of the research is to synthesize anticancer metabolites from a novel isolated strain of Streptomyces sp. from cultivated soil. In addition, the chemical characterization and identification of the produced active metabolite has been studied. The antimicrobial and cytotoxic potencies of EtOAc extract of S. rochei were evaluated. The extract inhibited the growth of Staphylococcus aureus, S. aureus (MRSA), Pseudomonas aeruginosa, Aspergillus fumigatus, Fusarium solani and Penicillium chrysogenum with inhibition zones (mm) of 16, 16, 17, 16, 22, 23, respectively. Furthermore, the extract exhibited noteworthy cytotoxic properties against the hepatoblastoma cell line (HepG2) and human epithelial cell line (Caco-2) with IC50 values of 818.10 μg/ml and 572.50 μg/ml, respectively. Based on the 16S rRNA sequencing, the bacterial strain was recognized as S. rochei both phenotypically and genotypically. It was then entered into the GenBank database under the accession number OR492299. The primary GC-MS chemical fingerprint of the EtOAc extract was characterized. Nineteen compounds were found, with relative amounts of 73.43, 5.61, and 3.13%, classified as the principal ingredients. These compounds included 1,3-benzenedicarboxylic acid, bis(2-ethylhexyl) ester, 13-docosenamide, (Z)-, and 9-octadecenamide, (Z). These results suggested that S. rochei EtOAc extract could be a valuable source of anticancer agents.