The aim of this study was to evaluate the effects of food hygiene training of food handlers on sanitary-hygienic conditions in hospital kitchens, based on microbiological analysis of smears taken in hospital kitchens. The study was conducted in the 1995-2009 period at the Clinical Centre Nit, Serbia. The food hygiene training was conducted in February 2005, by an infection control officer. 1,076 smears in the central kitchen and 4,025 smears in distributive kitchens were taken from hands and work clothes, work surfaces, equipment, and kitchen utensils. Microbiological analysis of smears was carried out in an accredited laboratory of the Public Health Institute Nit (Serbia). A significantly lower percentage of smears with isolates of bacteria (p<0.001) taken from hands and work clothes, work surfaces, equipment and kitchen utensils in the central and distributive kitchens was observed in the period following the food safety education programme (2005-2009). The most commonly isolated bacteria was: Enterobacter spp., Acinetobacter spp., Citrobacter spp., and E. coli. Our results confirmed that food hygiene training improved hygiene and is also an important component for the prevention of nosocomial infection.