The aim of this randomized, controlled, three-arm parallel-group, double-blinded clinical trial was to investigate the clinical and microbiological effects of the ozone application in stepwise excavation of primary molars.This study was conducted in in vivo conditions with 105 lower primary molars that had deep caries lesions. The teeth were randomly divided into three groups: (I) control group, conventional stepwise excavation with no disinfectants; (II) CHX (positive control) group, 2% chlorhexidine digluconate; and (III) experimental group, ozone application. Dentine samples were collected at 4 different time points (T0, at the first appointment, after partial removal of carious dentin; T1, at the first appointment, after applying disinfection procedure; T2, at the second appointment - at the end of the 4-month period - immediately after the removal of the temporary restoration; T3, at the second appointment, after the final excavation) for the microbiological analysis of mutans streptococci and lactobacilli and the total number of colony-forming units. Clinical changes including dentin color, humidity, and consistency were recorded. Data were analyzed using the Mann-Whitney U test, Friedman test, and chi-squared test (p = 0.05).The dentin became harder and drier after 4 months in all the groups. There was a gradual reduction in the total number of microorganisms in all the three groups. Bacterial reduction was greater in the CHX group compared to the ozone group (p = 0.002) and was greater in the ozone group compared to the control group (p = 0.015) after 4 months (control, 78%; CHX, 93%; ozone, 82%).Stepwise excavation of primary teeth provided successful outcomes in all the groups. Moreover, CHX, due to its practicality and cost-effectiveness, can be effectively used with one- or two-step indirect pulp therapies.Usage of cavity disinfectants in the stepwise excavation procedure contributes to the reduction of bacterial population in the cavity, which may allow the avoidance of the second step of the indirect pulp therapy for primary teeth.