Istamycins (ISMs) are 2-deoxyfortamine-containing aminoglycoside antibiotics (AGAs) produced by Streptomyces tenjimariensis ATCC 31603 with broad-spectrum bactericidal activities against most of the clinically relevant pathogens. Therefore, this study aimed to statistically optimize the environmental conditions affecting ISMs production using the central composite design (CCD). Both the effect of culture media composition and incubation time and agitation rate were studied as one factor at the time (OFAT). The results showed that both the aminoglycoside production medium and the protoplast regeneration medium gave the highest specific productivity. Results also showed that 6 days incubation time and 200 rpm agitation were optimum for their production. A CCD quadratic model of 17 runs was employed to test three key variables: initial pH, incubation temperature, and concentration of calcium carbonate. A significant statistical model was obtained including, an initial pH of 6.38, incubation temperature of 30 ˚C, and 5.3% CaCO3 concentration. This model was verified experimentally in the lab and resulted in a 31-fold increase as compared to the unoptimized conditions and a threefold increase to that generated by using the optimized culture media. To our knowledge, this is the first report about studying environmental conditions affecting ISM production as OFAT and through CCD design of the response surface methodology (RSM) employed for statistical optimization. In conclusion, the CCD design is an effective tool for optimizing ISMs at the shake flask level. However, the optimized conditions generated using the CCD model in this study should be scaled up in a fermenter for industrial production of ISMs by S. tenjimariensis ATCC 31603 considering the studied environmental conditions that significantly influence the production proces.