Research objectives: Biofilm of multidrug-resistant organisms and contamination of the hospital environment with biofilm is a challenge in the implementation of disinfection as infection prevention and control measure. The aim of this research was to examine the effect of gaseous ozone as a potential alternative disinfectant on the biofilm of multidrug-resistant organisms from the hospital environment as model organisms. Methods: In vitro part of the research included examining of the effect of gaseous ozone on the 24-hour biofilm of K. pneumoniae, A. baumannii and S. aureus on ceramics and polystyrene with the presence of different organic load. The physico-chemical characteristics of the material and the surface of the model organisms were examined. The parameters of the biocidal effect of gaseous ozone included the total number of cultivable bacteria, the total number of viable bacteria using ATP bioluminescence and Dead/Live staining and the effect on the total biomass using crystal violet staining. The effect on the total number of reactive oxygen species and morphological changes on the biofilm were monitored using digital microscopy, atomic force microscopy and scanning electron microscopy. The in situ effect of gaseous ozone was monitored on the biofilm of selected standard strains of model organisms in the dr. Ivo Pedišić General Hospital, Sisak, with accompanying monitoring of the microbiological cleanliness of the surfaces and the number of bacteria in the air. Results: Gaseous ozone in the concentration of 25 ppm during 1 hour of exposure causes a statistically significant reduction of the total number of cultivable and viable bacteria and a significant reduction of the total biomass in the biofilm on both materials and different organic load. The most sensitive model organism to the effect of ozone on both materials and with different types of organic load was A. baumannii, the least sensitive was S. aureus. Gaseous ozone causes a statistically significant increase in reactive oxygen species in all model organisms and morphological changes in the biofilm and individual bacterial cells in the sense of cell deformation and deposition on the cell membrane and cell destruction. Conclusion: Gaseous ozone is an effective disinfectant, however, it does not manage to completely remove biofilm of model organisms from ceramics and polystyrene on its own, and for successful biofilm removal, mandatory regular mechanical cleaning and washing before ozone disinfection in combination with another disinfectant is recommended.