The rhizosphere of plant possesses important microflora, which secretes wide chemical compounds including secondary metabolites necessary for plant growth and development. The microbial flora of alfalfa plant rhizosphere soil region was explored for functional activity and we found upto ten different pigmented colonies. Due to good functional diversity, this yellow pigmented colony was taken for further studies. Thus, the culture was molecularly characterized and identified for potent bioactive components responsible for antimicrobial activity. The selected culture mass was cultured and secondary metabolites were produced and extracted using ethyl acetate and subjected to GC-MS analysis. The antimicrobial study revealed selective activity against Streptococcus pneumonia, and Proteus sp with zone of inhibition to be 18 and 20 mm respectively.  Molecular identification of the isolate by 16S rRNA sequencing showed the isolate as Macrococcus equipercicus with 100 % similarity. Based on GC-MS analysis report 25bioactive compounds were identified and 13-docosenamide, hexadecanoic acid esters and quercetin were found in ethyl acetate extract. Conclusion: Thus the yellow pigmented gram positive cocci M.equipercicus isolated from Medicago sativa possessed wide antibacterial activity due to presence of quercetin. Through the studies, we were able to identify potent antibacterial compound producing bacteria from M. sativa plant rhizosphere soil.

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