The aim of this study was to evaluate in vitro antibacterial effect of 17 mixtures additives in various concentrations, specifically medium-chain fatty acids (MCFAs), myristic acid, cinnamic acid and citric acid, on selected bacteria, especially Staphylococcus aureus (ATCC 29213, ATCC 43300), Salmonella enteritidis (147/7F4), Salmonella typhimurium (K3), Enterococcus faecium (CCM 6226) and, Enterococcus cecorum (CCM 4285), by the standardized microdilution method in a 96-well microtitration plates. The minimum inhibitory concentrations (MICs) of tested additives were determined as the lowest concentration limiting the growth of bacteria in wells compared to a positive control of ≥80%. The additives were the least effective in inhibition of Gram-negative strains of Salmonella (S. enteritidis, MICs: 2048-≥4096 µg ml-1; S. typhimurium, MICs: ≥4096 µg ml-1). Both strains of S. aureus were sensitive to the presence of all additives. The lowest MIC for S. aureus ATCC 29213 was 56 µg ml-1 using C12/C14 (70/30). The remaining additives inhibited growth at concentrations 64-2048 µg ml-1. Interestly, S. aureus ATCC 43300 was the most sensitive to C12/C14+GML+CitA (24,5/10,5/35/30) at MIC 64 µg ml-1. Other additives showed MICs in range 64-2048 µg ml-1. Gram-positive bacteria, E. cecorum and E. faecium were the most sensitive to C12/C14 (70/30) (MIC 32 and 64 µg ml-1). The remaining values of MICs ranged from 128 to ≥4096 µg ml-1. Compared to Gram-negative bacteria, antibacterial effect of MCFAs, GML, citric and cinnamon acid was observed mostly to Gram-positive bacteria.