Disinfection of surfaces with various functional purposes is a relevant measure for the inactivation of microorganisms and viruses. This procedure is used almost universally, from water treatment facilities to medical institutions and public spaces. Some of the most common disinfectants the World Health Organization recommends are chlorine-containing compounds. Sodium and calcium hypochlorites are only used for disinfection of the internal surfaces of water treatment facilities. However, it is known that ozone is a more powerful oxidizing agent. This study compares the effectiveness of inactivating yeast-like fungi Candida albicans, Gram-positive Bacillus subtilis, and Gram-negative bacteria Escherichia coli with aqueous ozone and sodium hypochlorite solutions. This study used ozone solutions in water with a concentration of 0.5–1.5 mg/L and sodium hypochlorite solutions with an active chlorine concentration of 50–150 mg/L. Steel and polymeric plates were used as substrates. Comparison of the CT (concentration by time) criterion at the ratio of LD50 in NaClO to ozonated water shows that the smallest difference, around 100 times, was observed in the inactivation of Candida albicans. The maximum difference is up to 230 times in the inactivation of Bacillus subtilis.