Oral biofilm formation is the main reason for both caries progression and soft tissue diseases. Preventing the formation and promotion of biofilm has been known as the first attempt to prevent the development of caries and soft tissue problems in the oral cavity. The present study aimed to assess the effect of ozone and its combined use with chlorhexidine (CHX) and fluoride on the complex biofilm formation of pediatric patients under in situ conditions. Extracted bovine teeth were sterilized and cut into 2 × 3 mm 2 sections. The samples were placed in removable maxillary plates and 10 healthy individuals (6 boys, 4 girls; aged 7–14 years) were asked to wear these plates for 6, 24 and 48 hours. Afterwards, the tooth samples were removed, and anti-plaque agents were applied to the time-related plaque formation. Plaque thickness and viable bacterial percentages were detected by confocal laser scanning microscopy. All materials used in the study decreased the plaque formation and the percentage of viable microorganisms compared with the control group (physiological saline). In 6- and 24-hour biofilm evaluations, ozone-CHX was the most effective group in decreasing the plaque thickness ( P > 0.05). Ozone-CHX and Ozone-Fluoride groups were found to be better in 48-hour biofilm assessments in caries-free group ( P > 0.05). Ozone-CHX group showed a better inhibitory effect on the viability of microorganisms in 6-, 24- and 48-hour biofilm formations ( P < 0.05). Although CHX has been known as the gold standard for inhibiting the oral biofilm formation, according to the results of the study, gaseous ozone and its combined use with CHX have shown better results in reducing the biofilm thickness and viable bacterial percentages of in situ formed time-related biofilm formation in pediatric patients. The use of gaseous ozone can be preferred in clinical conditions in pediatric patients instead of the CHX agents.