Abstract: Background and Objective: With increase in multiple drug resistance pathogens, it is necessary to look for new drug study compounds of microbial origin. Thus study was aimed to identify the rhizosphere microflora of unexplored alfalfa plant for new antimicrobials. Materials and Methods: Based on screening done, the isolates were subjected to antibacterial activity against selected bacteria. The isolate was mass cultured and secondary metabolites were extracted using ethyl acetate. The crude extracts collected were subjected to FTIR and GC-MS analysis. Results: Based on functional diversity analysis, the isolate subjected to anti-bacterial activity revealed significant activity against Klebsiella and Staphylococcus aureus with zone of inhibition in the range of 17-18 mm. Based on GC-MS analysis reports, six compounds were identified and 11-Octadecanal responsible for bio-activity. FT-IR results showed that N-H stretching functional group dominantly present in the extract. Molecular identification of the isolate by 16S rRNA sequencing showed the isolate as Bacillus horikoshii. Conclusion: The study results showed that the isolate Bacillus horikoshii, Gram-positive spore forming bacteria had wide antibacterial activity due to 11-Octadecanal. Thus Alfalfa plant rhizosphere region harbors antibacterial potential microbes.