β-lactamase inhibitors are potent synergistic drugs to reverse the drug resistant populations to sensitive ones. The production of such commercially important compounds needs much consideration on its production from the source organism. In the present study, a marine Streptomyces derived β-lactamase inhibitory compound kalafungin was optimized under solid state fermentation conditions. This study intended to facilitate the production of β-lactamase inhibitor by applying statistical methods of optimizing the culturing process variables for secondary metabolite biosynthesis. The initial optimization revealed starch as a main carbon source played a vital role in kalafungin production at 0.1% NaCl concentration by one-factor-at-a-time method. Further it was carried forward to maximize the productivity of kalafungin through fractional factorial design and the optimum medium composition by Response Surface Methodology (RSM). Plackett-Burman design and Box-Behnken design experiments have given the maximum production of 51.4 mg/L of kalafungin at 29°C for 8 days of incubation which is 25.06% more than the unoptimized conditions. The experimental values are closer to predicted values and it has strongly supported the use of RSM in fermentation condition optimization for kalafungin production.