Antibiotic activity against various gram positivebacteria including Staphylococcus aureus and Enterococcuswas ascertained from a soil-isolated microbial strainStreptomyces sp. CS392. The antibiotic activity of the strainwas maximized by using a dual-stage, multivariate statisticaloptimization framework based on the response surfacemethodology considering a lab-scale fermentation process. Multiple nutrient constituents of the fermentation brothwere jointly optimized in the first stage, while the fermentationculture conditions were optimized in the subsequentstage. Based on the empirical models derived from the dualstagestatistical optimization framework, 39.79 % ofcumulative enhancement in the antibiotic activity wasobtained (analytically) at the concurrent optimal settings(Optimal nutrient composition for the first stage of optimization:29.82 glucose, 7.6 peptone, 4.678 MgCl2 and0.5005 g/l casamino acid; and optimal fermentation conditionfor the second stage of optimization: incubationperiod 47.55 h; incubation temperature 29.15 C; and pH8.36). The analytically depicted enhancement in the antibioticactivity was validated experimentally.