Short chain fatty acids (SCFAs) are commonly produced by healthy gut microbiota and they have a protective role against enteric pathogens. SCFAs also have direct antimicrobial activity against bacterial pathogens by diffusion across the bacterial membrane and reduction of intracellular pH. Due to this antimicrobial activity, SCFAs have promising applications in human health and food safety. In this study, the minimum inhibitory concentrations (MICs) of four SCFAs (acetic acid, butyric acid, propionic acid, and valeric acid) in Salmonella strains isolated from poultry were determined. The effect of subinhibitory concentrations of SCFAs in Salmonella biofilm formation, motility, and gene expression was also evaluated. Butyric acid, propionic acid, and valeric acid showed a MIC of 3750 µg/mL in all strains tested, while the MIC of acetic acid was between 1875 and 3750 µg/mL. Subinhibitory concentrations of SCFAs significantly (p < 0.05) reduced the motility of all Salmonella strains, especially in the presence of acetic acid. Biofilm formation was also significantly (p < 0.05) lower in the presence of SCFAs in some of the Salmonella strains. Salmonella strain. Salmonella Typhimurium T7 showed significant (p < 0.05) upregulation of important virulence genes, such as invA and hilA, especially in the presence of butyric acid. Therefore, SCFAs are promising substances for the inhibition of the growth of foodborne pathogens. However, it is important to avoid the use of subinhibitory concentrations that could increase the virulence of foodborne pathogen Salmonella.