ABSTRACTContamination withSalmonellaspp. andListeria monocytogenesis concerning across low-moisture food (LMF)-processing environments due to the pronounced survival of these organisms under dry conditions. This study treated desiccated bacteria with acetic acid delivered by oil with and without water-in-oil (W/O) emulsion. The influences of cellular desiccation, emulsion water concentration, water activity (aw), and treatment temperature were investigated. Acetic acid dissolved in oil (i.e., acidified oil) showed low levels of antimicrobial efficacy. After treatment with acidified oil (200 mM acetic acid at 22°C for 30 min),Salmonella entericaserovar Enteritidis phage type 30 cells desiccated to 75% equilibrium relative humidity (ERH) and 33% ERH were reduced by 0.69 and 0.05 log CFU/coupon, respectively. The dispersion of a low level of water (≥0.3%, vol/vol) within the acidified oil with the surfactant (i.e., acidified W/O emulsion) significantly enhanced the antimicrobial efficacy. After treatment with the acidified W/O emulsion (200 mM acetic acid at 22°C for 20 min), desiccatedSalmonella(4-strain cocktail) andL. monocytogenes(3-strain cocktail) cells were reduced by >6.52 log most probable number (MPN)/coupon, regardless of the desiccation levels. Increased efficacy was observed with temperature elevation. Reduced efficacy was observed when glycerol was added to the aqueous phase of the emulsion to decrease the solutionaw, indicating that the enhanced efficacy of the acidified W/O emulsion was associated with differential osmotic pressure. The antimicrobial mechanism may be due to the membrane disruption induced by acetic acid, in combination with the hypoosmotic stress provided by W/O emulsion, creating cellular lysis, as illustrated by electron micrographs.IMPORTANCEAqueous-based cleaning and sanitation are undesirable in processing facilities that manufacture low-moisture foods such as peanut butter and chocolate. Alcohol-based sanitization is advantageous because it leaves no residue on the contact surface but requires the processing facility to close temporarily due to flammability. At >6.52 log kill of desiccatedSalmonellaandListeria monocytogenescells, the developed oil-based formulation has the potential to be an effective dry sanitation method.