Microemulsion is colloidal dispersion of oil and water having droplet size in the range of 2-50 nin and is stabilized by the interfacial layer of surfactant. Antibacterial application of microemulsion in prevention of wound in-fection is not much explored. Previous work by our group has demonstrated the role of surfactant concentration in determining the droplet diameter, optical property and the stability of microemulsion. Also, the formulated microemulsion triggered the wound healing process in wistar rats. In the present work, selected microemulsion formulation (CMF4 : droplet size = 5.79 nm; oil/surfactant/water composition = 6/24/70 (vol/vol)) demonstrated bactericidal activity against wound isolate Macrococcus caseolyticus (confirmed by 16S rDNA sequencing). Inactivation kinetics exhibited time and dose dependent killing of Macrococcus caseolyticus by CMF4 microemulsion. Complete loss of Macrococcus caseolyticus viability was observed after incubating with 10-fold diluted CMF4 microemulsion formulation for 20 min. The CMF4 microemulsion also exhibited antibacterial activity at higher dilution of 1000-fold. FTIR study illustrated alteration of functional groups of Macrococcus caseolyticus surface upon CMF4 microemulsion treatment. Scanning electron micros-copy images illustrated the morphological changes in Macrococcus caseolyticus after interaction with CMF4 microemulsion. The outcome of the presented study suggested CMF4 microemulsion may be applied for prevention of infection in wound management. © 2015 Scientific Publishers. All Rights Reserved.