Hospital-acquired infections represent one of the most important challenges in medicine in the 21st century, and multi-resistant microorganisms stand out as their main causative agents. Among them is Klebsiella pneumoniae, an emerging and pathogenic bacterium that can contaminate the hospital environment in the form of a biofilm, whose eradication with standard disinfectants is very difficult. Therefore, in recent years, new modern biofilm suppression technologies have been developed with the aim of greater microbiological purity. One of the most effective methods is the application of gaseous ozone since ozone has strong antimicrobial properties and a high oxidation potential. The aim of this work was to determine the effectiveness of gaseous ozone in reducing the number of bacteria in the biofilm of five clinical and one standard strain of K. pneumoniae during a 60-minute exposure to ozone and to quantify the amount of biofilm before and after treatment using different methods. Biofilms of all tested strains were created on ceramic plates measuring 2.5 cm x 2.5 cm for 24 hours, which were then exposed to ozone for 1 hour. After washing, the total number of cultivable bacteria was determined, biological activity and total biomass were measured after staining with crystal violet. Analysis with a scanning electron microscope visualized the destruction of the biofilm. Gaseous ozone proved to be very effective and statistically significant in destroying the created bacterial biofilms on ceramic tiles. Ozone treatment caused a decrease in the total number of cultivable bacteria to 2.63log10CFU/cm2, followed by a decrease in total biomass to 67.17%. The inhibition percentages varied from 36.61% to 99.76%, depending on the quantification method used. All methods used in this research are effective in determining the ability of gaseous ozone against biofilms, but additional research is required.