Natural products from plants have been in therapeutic use since many years. Novel molecules from plant sources have been isolated, characterized and used in managing different diseases. Alkannatinctoria (Family: Boraginaceae) is an important herb stated in Siddha medicine, which is used to cure many ailments alone and in formulations. The present study comprises isolation of Acetyl Alkannin, from hexane extract of the root bark of A. tinctoria, through silica gel column chromatography. Structure of the compound was elucidated through MS and H1, C13 NMR. The pure compound was analyzed for its antimicrobial action against common human pathogens, where it showed considerable Minimum Inhibitory Concentrations (MIC) against most of the pathogens. Significant activity was observed against Bacillus subtilis (MIC: 28 μg/ml), Enterococcus faecalis (MIC: 28 μg/ml), Escherichia coli (MIC: 12 μg/ml), Pseudomonas aeruginosa (MIC: 12 μg/ml). Studies on its combination with other commercial antibiotics depicted synergistic patterns against P. aeruginosa with most of the antibiotics. Also, it worked both synergistically and additively against microbes with other antibiotics. Thus, it could be used in combination with other antimicrobial agents in managing critical microbial infections.