ABSTRACT: A potent actinobacterial strain isolated from the marine samples of Bheemunipatnam beach, Visakhapatnam, India, was identified as Rhodococcus sp. VLD-10 using the conventional and genomic (16S rRNA) approaches. Bioactive compounds responsible for the antimicrobial activity of the strain were elucidated by cultivating the strain VLD-10 in a modified yeast extract-malt extract-lactose broth followed by subsequent chromatographic and spectroscopic analyses. Extraction, purification, and structural confirmation of five compounds, viz., benzoic acid, 2-nitrobenzaldehyde, 4-chlorobenzaldehyde, nonadeconoic acid, and 3-isopropylhexahydro-1H-pyrido[1,2-a] pyrazine-1,4(6H)-dione, from Rhodococcus sp. VLD-10 were fruitfully described. The bioactivity of the compounds isolated from the strain VLD-10 against Gram-positive as well as Gram-negative bacteria, yeast, and molds was tested and their minimum inhibition concentration was reported. Antibacterial activity of 3-isopropylhexahydro-1H-pyrido[1,2-a] pyrazine-1,4(6H)-dione is more prominent against Bacillus subtilis, B. cereus, B. megaterium, Corynebacterium diphtheriae, and Escherichia coli, whereas its antifungal spectrum showed less potency against yeast and fungi. This is the first report on the natural occurrence and bioactivity of 3-isopropylhexahydro-1H-pyrido[1,2-a] pyrazine-1,4(6H)-dione from Rhodococcus sp. VLD-10.