Lemongrass (Cymbopogon) and Khus (Vetiver) oil, an essential oils play an important role in antibacterial and anti-inflammatory properties. Present study was designed for the in-vitro efficacy of essential oils, Lemongrass and Khus oil, against common mastitis causing organisms namely Klebsiella spp. and E.coli. Total 220 animals were screened using California mastitis test and white side test in the study area. Out of 200 animals total 124 animal were found positive for sub clinical mastitis. Of these samples, around 30 samples were subjected to further bacterial isolation, wherein, E.coli and Klebsiella spp. were isolated. The best combination of oils was further tested as a spray in lactating buffaloes suffering from SCM. 100% of E.coli isolates were sensitive to Ceftriaxone, Enrofloxacin, Ceftriaxone and Sulbactam combination followed by Streptomycin and Gentamicin (90%), Vancomycin and Tetracycline (60%), Oxytetracycline (40%) and Penicillin (20%). The order of sensitivity observed against Klebsiella spp. was Enrofloxacin and Gentamicin (100%), Streptomycin, Ceftriaxone & Sulbactam (90%), Ceftriaxone (70%), Oxytetracycline, Amoxycillin (50%), Penicillin, Vancomycin and Tetracycline (40%). Enrofloxacin had the maximum activity against S. aureus and Klebsiella spp., whereas, ceftriaxone has maximum efficacy against E.coli. Overall, maximum zone of inhibition was achieved by 75% Lemongrass Oil + 25% Khus Oil combination across the two bacterial isolates categories. Further, the efficacy of essential oil of Cymbopogon citratus and Vetiveria zizanoides as a teat spray (75% lemongrass oil + 25% khus oil) combination in prevention of mastitis was studied. This was tested in healthy buffaloes as well as SCM affected buffaloes. The animals were divided in 4 groups for the study of the efficacy of essential oil combination as a teat spray with 10 animals in each group. Post 15 days of therapy, Group 1 (healthy buffaloes) had minor changes in evaluated parameters. Group 2 (healthy buffaloes) showed improved results where the healthy buffaloes were sprayed with essential oil spray. Group 3 (SCM buffaloes) where no treatment was given, an increase in SCC was observed. The final group 4 animals (SCM buffaloes+ spray) showed 70% recovery with a decline in CMT scores, WST scores as well as SCC scores.