Although its wide utilization in microbial cultures, the one factor-at-a-time method, failed to find the true optimum, this is due to the interaction between optimized parameters which is not taken into account. Therefore, in order to find the true optimum conditions, it is necessary to repeat the one factor-at-a-time method in many sequential experimental runs, which is extremely time-consuming and expensive for many variables. This work is an attempt to enhance bioactive yellow pigment production by Streptomyces thinghirensis based on a statistical design. The yellow pigment demonstrated inhibitory effects against Escherichia coli and Staphylococcus aureus and was characterized by UV-vis spectroscopy which showed lambda maximum of 449. The FTIR and GC-MS analysis showed that the colorings in this type of product are due to the presence of chromo peptides. Furthermore, the GC-MS measurement determined the presence of 4 compounds, as it gave 4 different retention times within this yellow pigment, but with different percentages, except for the compound BHT when the retention time was 17.86 minutes. Starch casein broth (SCB) was selected as an optimized medium for yellow pigment production. The optimization process was first started with one factor at time method, revealing that maltose and casein were the best carbon and nitrogen sources. Response surface methodology based on central composite design was conducted to obtain the optimal combinations of maltose and casein concentrations, pH, and inoculum size for maximum production of yellow pigment. The results showed that casein was the most effective parameter with F-value 393.1 and the model exhibited good fitting with a correlation coefficient of 0.946. Moreover, the actual maximum yellow pigment product 0.80 nm which aggregated with a predicted value 0.835 nm at maltose concentration 8 g/L, casein 5 g/L, KNO3 0.01 g/L, pH 6 and inoculum size 5%.