Microemulsion was formulated using mustard oil, non-ionic surfactant Tween 20 and water. MF5 formulation showed higher stability with droplet size in the range of 118-430nm with a polydispersity index of 0.236. Low polydispersity index confirms the homogeneity of emulsion formulation. Mustard oil based microemulsion formulation demonstrated effective antibacterial activity against E. coli. Kinetics of killing of bacterial polutaion showed that 10-fold diluted MF5 formulation was effective in 5 log reductions of E. coli population in an interaction period of 60 min. Alteration in bacterial surface functional groups were observed in microemulsion-treated cells. These results pertaining to FTIR of untreated and treated E. coli substantiated the kinetics of killing of bacteria upon microemulsion treatment.