Streptomyces(S.) rimosus NRRL 2455 produces paromomycin, a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic (2DOS-ACAGA) with broad-spectrum activity against most of the Gram-positive and Gram-negative bacteria as well as protozoa. The mutation has become one of the beneficial methods used in enhancing the microbial production. Improvement of the paromomycin production by S. rimosus NRRL 2455 was achieved via irradiation mutagenesis using gamma (ˠ) radiation. The culture of S. rimosus was irradiated with different doses of gamma radiation (3, 4 and 5 KiloGray (KGy) to find out the best dose for the mutation that gave 99.99% killing. The optimum dose was found to be 4 KGy. Six morphologically changed colony types appeared on tryptic soy agar plates. These colonies were bio-assayed for their antimicrobial activity against standard Staphylococcus aureus ATCC 25923 using agar well diffusion technique. A mutant coded 5M showed about 1.44, and 2 fold increase in its activity as compared with the wild-type when cultivated in basal culture or optimized media (soybean meal 30 g/L, NH₄CL 4 g/L, CaCO₃  5 g/L and glycerol 40 ml/L), respectively. Moreover, high genetic stability was observed upon subsequent culturing of 5M-mutant. Therefore, S. rimosus mutant-5M can be used as a potential industrial strain for paromomycin production.