AIM: To assess the microbial effect of ozone gas on dentinal lesions in young permanent molars using the stepwise excavation. STUDY DESIGN: An experimental, controlled clinical trial was performed. The sample included 80 immature first permanent molars, showing deep occlusal carious cavities that were indicated for stepwise excavation. Following first step of dentin excavation, the sample was divided into test (ozone gas) and control (calcium hydroxide (Ca(OH)) base material) groups. One half of the cases in each group were evaluated for microbiological changes after 6 months, and the other half after 12 months. RESULTS: Mutans streptococci (MS), Lactobacilli, and Candida counts were significantly reduced immediately after ozone application in the test group (P ≤0.05). At the final assessment period, MS and Lactobacilli were significantly reduced in the test group (P ≤0.05). Meanwhile, the Candida counts were significantly reduced only in the test group of the 6 and 12 month-cases (P ≤0.05). Regarding the control group, the significant reduction in microbial count was observed with MS after 6 and 12 months (P ≤0.05). No significant differences were observed between test and control groups at different evaluation periods (P >0.05). CONCLUSIONS: Ozone gas had a significant antimicrobial effect in deep class I carious lesions.