Cleaning and disinfection have big importance through the production, manufacturing and distribution of food. With proper cleaning method and selection of disinfectants we can effectively prevent contamination of foods with unwanted microorganisms. Within the diploma work we investigated antibacterial activity of chlorhexidine diacetate. The activity was evaluated by determining minimum inhibitory concentration (MIC) for different bacteria, as follows, Listeria monocytogenes (as gram-positive bacterium), Staphylococcus aureus (as toxigenic bacterium), Bacillus cereus (as sporogenic bacterium), Escherichia coli and Salmonella enterica (as gram-negative bacteria). MIC was determined by micro-dilution method after adding INT reagent (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chloride) that dyed metabolic active bacterial cells. The number of surviving bacteria was also determined with the plate count method. Chlorhexidine diacetate was the most efficient for gram-positive Staph. aureus and L. monocytogenes stains, where the MIC was 0.9±0.3 mg/l. The gram-negative E. coli strain had MIC of 1.1±0.3 mg/l. The gram-negative S. enterica strain had the highest MIC of 8.7±2.5 mg/l. Sporogenic B. cereus had MIC of 1.5±0.6 mg/l. The hypothesis that gram-negative bacteria such as E. coli and S. enterica are more resistant to chlorhexidine diacetate than gram-positive bacteria such as Staph. aureus, B. cereus and L. monocytogenes was confirmed. Sporogenic bacteria like B. cereus were more resistant than non-sporogenic gram-positive bacteria. Usage of appropriate concentrations of chlorhexidine decrease the presence of bacteria.