Optimization of the fermentation medium components for maximum gentamicin production by Micromonospora echinospora ATCC 15838 was carried out. Response surface methodology was applied to optimize the medium constituents. A 2(4) full-factorial central composite design was chosen to explain the combined effects of the four medium constituents, viz. starch, soyabean meal, K2HPO4, and CoCl2 and to design a minimum number of experiments. A second order model was developed and fitted using least square method. The R2 value of the model was 0.9723, which shows that model is best fit for the present studies. The results of analysis of variance and regression of a second order model showed that the linear effects of starch (p < 0.001697) and CoCl2 (p < 7.99E-13), and cross product effects of starch and soyabean meal (p < 0.029876) and soyabean meal and CoCl2 (p < 0.008909) were more significant, suggesting that these were critical variables having the greatest effect on the production of gentamicin in the production medium. The optimized medium consisting of 9 g/L starch, 3 g/L soyabean meal, 0.9 g/L K2HPO4, and 0.01 g/L CoCl2 predicted 850 mg/L of gentamicin which was almost 110% higher than that of the unoptimized medium. The amounts of starch, soyabean meal, and K2HPO4 required were also reduced with RSM.