Due to emergence of drug resistant pathogens, nearly all available medicines are becoming ineffective against these life threatening pathogens so there is dire need for the discovery of compounds having unique modes of action. During our previous studies, actinomycetes designated as 196 and RI.24 were isolated, screened for bioactive compounds production and characterized using 16S rRNA gene sequencing. Colony 196 was identified as strain of Streptomyces albolongus (100% sequence similarity) and RI.24 as strain of Streptomyces enissocaesilis (100% sequence similarity). In current study, potential bioactive compounds produced by these strains were characterized. Cold extraction method was applied for taking out of bioactive compounds from actinomycetes. Minimum inhibitory concentration (MIC) determination of compounds from these strains showed activity nearly in the range of commercial antibiotics (strain 196 0.0075 mg/ml, RI.24 0.25 mg/ml and chloramphenicol 0.0075 mg/ml, ampicillin 0.025 mg/ml). Structural elucidation of these compounds was carried out using spectroscopic techniques of LC MS/MS and H-1 NMR. Compounds K-252-C-Aglycone, indolocarbazole alkaloid, decoyinine, cycloheximide were detected from strain 196 whereas daunorubicin, hygromycin B, agecorynin F, indinavir-N-glucuronide and minocycline were identified from strain RL24.Current study reports these compounds for the first time from strains of Streptomyces albolongus and Streptomyces enissocaesilis. Present investigation also suggests that strains 196 and RI.24 contain polyketide synthase-I (PKS-I) and non-ribosomal peptide synthetase (NRPS) gene clusters which are responsible for the production of bioactive compounds. The results of this study can be used by the scientific world or pharmaceutical industries for the development of new drugs/formulations by applying more advanced techniques.