Insecticide resistance in agricultural pests has prompted the need to discover novel compounds with new modes of action. We investigated the potency of secondary metabolites from seventy endophytic actinobacteria against laboratory and field strains ofSpodoptera littoralis(fourth instar), comparable to the bioinsecticide spinetoram (Radiant SC 12%). Endophytes fromArtemisia herba-albaandA. judaicawere highly effective. Chemical profiling of the most potent metabolite of the strainStreptomycessp. ES2 was investigated using LC-QTOF-MS-MS technique, and the activity was validated through molecular docking studies. Metabolic extracts from actinobacteria belonging toStreptomyces,Nocardioides, andPseudonocardiashowed immediate and latent death to theSpodoptera littoralisfourth instar larvae. The metabolite from strain ES2 has shown the most promising and significant histopathological and inhibitory effects on the fourth instar larvae. ES2 metabolite caused lesions in the body wall cuticle, indicating a different mode of action than that of Radiant. Chemical profiling of ES2 showed the presence of cyromazine (molt inhibitor), 4-nitrophenol, and diazinon as key constituents. In conclusion, these findings suggest that secondary metabolites from endophytic actinobacteria inhabiting wild medicinal plants can be a sustainable source for promising natural biocontrol agents. This is the first illustration of the insecticidal activity ofArtemisiaspp. microbiome, and natural cyromazine synthesis by actinobacteria.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13568-023-01550-x.