Streptomyces sp. RAB12 having potential to produce novel actinomycin group compounds was isolated from soil samples collected from CSIR-Indian Institute of Chemical Technology, Hyderabad, India, garden premises using International Streptomycetes Project (ISP) protocols. The 16S rRNA sequence of the strain RAB12 exhibited identity with Streptomyces sp. 13647M and the sequence was deposited in NCBI under the accession number KY 203650 while the strain RAB12 was deposited in The Microbial Type Culture Collection and Gene Bank (MTCC) with accession number MTCC 12747. Cell-free extract of this novel strain revealed two bioactive principles viz., RSP 01 and RSP 02. HR-MS analysis indicated a molecular mass of 1269.61 and 1270.63 m/z g/mol for RSP 01 and RSP 02, respectively. Proton H-1, C-13 NMR, 2D NMR and mass spectroscopy analysis revealed a similar fingerprint to that actinomycin D except for a peak at delta H3.59 J (H-1 NMR) and delta 208.88 (C-13 NMR) for RSP 01 compound suggesting the presence of keto carbonyl at 5-oxo position on the proline moiety which is absent in actinomycin D. Purified RSP 02 depicted a similarity with RSP 01 except a peak in the H-1 proton NMR at delta H 3.81 J. HR-ESI mass spectra confirmed the molecular formulae for RSP 01 and RSP 02 as C62H84N12O17 and C62H86N12O17, respectively. Antimicrobial activity profile revealed higher antimicrobial activity against bacterial strains (Pseudomonas aeruginosa, Micrococcus luteus, Staphylococcus aureus, Salmonella typhi, and Bacillus subtilis) and Candida albicans compared to standard actinomycin D. MIC and MBC for RSP 01 were observed to be 0.0039 and 0.0078 (mu g/ml) against C. albicans, while for actinomycin D, it was found to be 0.031 and 0.62 (mu g/ml), respectively indicating a tenfold higher potency. Thus, these RSP 01 and RSP 02 compounds from Streptomyces sp. RAB12 may be promising candidates for industrial and clinical applications.