Weak organic acids are widely used as preservatives and disinfectants in the food industry. Despite their widespread use, the antimicrobial mode of action of organic acids is still not fully understood. This study investigated the effect of acetic acid on the cell membranes and cellular energy generation of four Salmonella strains. Using a nucleic acid/protein assay, it was established that acetic acid did not cause leakage of intracellular components from the strains. A scanning electron microscopy study further confirmed that membrane disruption was not the antimicrobial mode of action of acetic acid. Some elongated Salmonella cells observed in the micrographs indicated a possibility that acetic acid may inhibit DNA synthesis in the bacterial cells. Using an ATP assay, it was found that at a neutral pH, acetic acid caused cellular energy depletion with an ADP/ATP ratio in the range between 0.48 and 2.63 (p<0.05) that was apparent for the four Salmonella strains. We suggest that this effect was probably due solely to the action of undissociated acid molecules. The antimicrobial effect of acetic acid was better under acidic conditions (ADP/ATP ratio of 5.56 ± 1.27; p<0.05), where the role of both pH and undissociated acid molecules can act together. We concluded that the inhibitory effect of acetic acid is not solely attributable to acidic pH but also to undissociated acid molecules. This finding has implication for the use of acetic acid as an antimicrobial against Salmonella on food products, such as chicken meat, which can buffer its pH.