Pest control by biological means is an effective, eco-friendly, and promising method that typically involves compounds naturally derived from actinomycetes. Thus, the present study aimed to screen, characterize, and identify the structure of insecticidal compounds from Streptomyces sp. KR0006 and increase the activity through mutagenesis. In the examination of the insecticidal activity level of the isolates, Streptomyces sp. KR0006 metabolite showed significant activity against larvae and moths of Plutella xylostella. Taxonomic analyses of the 16S rRNA gene sequences revealed that the isolated KR0006 strain tended to be 99% consistent with Streptomyces cinereoruber strain NBRC 12756. Three active compounds isolated from the culture filtrate of KR0006 were purified by solvent partition, mid-pressure liquid chromatography (MPLC), Sephadex LH20 column chromatography, and high-performance liquid chromatography (HPLC). By performing 1H-NMR, 13C-NMR, and 2D-NMR experiments, and high-resolution electrospray ionization mass spectrometry analysis, the 316-HP2, 316-HP3, and 316-HP5 compounds were inferred as antimycin A3a (MW, 519.; C26H36N2O9), antimycin A8a (MW, 534; C27H38N2O9), and antimycin A1a (MW, 548; C28H40N2O9) respectively. Mutant U67 obtained from exposure to ultraviolet (UV) irradiation (254 nm, height 17 cm) for 70 seconds resulted in a 70% more larval mortality than that of the initial wild culture. The second mutation of the culture broth enhanced insecticidal activity by 80 and 100% compared with the first mutation and initial medium, respectively. Our study found that Streptomyces sp. KR0006 strain produces insecticidal active compounds and could be used for practical pest management.