Streptomyces, the dominant species of actinomycetes, contribute to a large proportion of bioactive natural products. In our previous study, over 2400 culture extracts were subjected to bioassays against Candida albicans and Staphylococcus aureus. Among these, Streptomyces javensis named Inha503, was found to produce nigericin, which exhibits antimicrobial activity against gram-positive bacteria, such as Micrococcus luteus and S. aureus. This study aimed to understand the genomic features of S. javensis Inha503 and optimize the downstream process to improve the titer of nigericin derivatives. Identification of the nigericin biosynthetic gene cluster (BGC) in the chromosome was confirmed using whole-genome bioinformatics and core BGC knockout results. The cultivation time was optimized to 10 days, and nigericin derivatives were identified and quantified using LC/MS. Among the seven-production media for Streptomyces, the highest production of nigericin was obtained in the R5 medium, which exhibited a 1.75-fold increase compared to the previous conditions. Additionally, the nigericin extraction process was optimized using ethyl acetate as a single solvent and a mixture of ethyl acetate and ethanol in a 7:3 ratio. The highest titer of nigericin derivatives were obtained in R5 medium after 10 days with extraction using a mixture of ethyl acetate and ethanol, implying that optimized purification processes with genetic insights of S. javensis Inha503 as a promising platform for bioactive compound production such as nigericin.