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