Background The application of disinfectants on dental instruments is one way to prevent cross-infection. Cross infection can occur due to microorganisms found in blood, saliva and dental plaque which can contaminate the instruments used by dental health workers. Thus, indirect contact transmission of pathogenic microorganisms occurs from instruments that have been contaminated by dental health workers. Ozone water and 4.8% chloroxylenol are disinfecting agents used to disinfect medical instruments. This study aimed to determine the effectiveness of disinfection of ozone water and 4.8% chloroxylenol in reducing the number of bacterial colonies on dental extraction instruments at the USU Dental and Oral Hospital between October-December 2022. Methods The samples used were mandibular molar pliers that have been used in tooth extraction procedures. This study was experimental and used three sample groups, where each group consisted of 10 tooth extraction instruments. The treatment group used ozone water and 4.8% chloroxylenol and the negative control group was cleaned with distilled water. The test effectiveness in this study used bacteria colony counter using the scatter cup method. Data were analyzed using the Kruskal Wallis and Mann-Whitney U tests. Results The results of the data analysis showed a p-value ≤0.001, which means that there was a significant difference in the disinfection using ozone water and 4.8% chloroxylenol on the number of bacterial colonies on dental extraction instruments. The results of this study show that the average number of bacterial colonies formed in the ozone water treatment group was 4.00 ± 4.32, 16.00 ± 6.65 in the 4.8% chloroxylenol treatment group, and 217.50 ± 39.24 in the negative control group (Aquadest). Conclusions From this study it can be said that ozone water is more effective in disinfecting dental extraction instruments than 4.8% chloroxylenol.