Aims This study was performed to evaluate the efficacy of butanoic acid against bacterial pathogens including Acinetobacter baumannii and Staphylococcus pseudintermedius. Methods and Results Vegetative bacteria were exposed to butanoic acid in vitro and log reduction was quantified using viable count assays. The maximum (8 and 9) log inactivation was determined by qualitatively assaying for growth/no-growth after a 48-h incubation (37 degrees C). Membrane integrity after exposure to butanoic acid was determined by propidium iodide staining, scanning electron microscopy, membrane depolarization and inductively coupled plasma analysis. Cytosolic pH was measured by 5-(6-)carboxyfluorescein succinimidyl ester. Conclusions Inhibitory concentrations of butanoic acid ranged between 11 and 21 mmol l(-1) for Gram-positive and Gram-negative species tested. The maximum log reduction of A. baumannii was achieved with a 10-s exposure of 0 center dot 50 mol l(-1) of butanoic acid. Staphylococcus pseudintermedius required 0 center dot 40 mol l(-1) of butanoic acid to achieve the same level of reduction in the same time period. Inactivation was associated with membrane permeability and acidification of the cytosol. Significance and Impact of the Study: Antibiotic resistance among bacterial pathogens necessitates the utilization of novel therapeutics for disinfection and biological control. These results may facilitate the development of butanoic acid as an effective agent against a broad-spectrum of antibiotic-resistant bacterial pathogens.