The purpose of this clinical study was to investigate the types of microorganisms in deep proximal caries lesions and the efficacy of elimination of microorganisms after a 24 s ozone application to proximal cavity lesions prepared for restoration. Sixteen caries-active volunteers (female: 8, male: 8; age range: 35-55), with proximally situated deep caries lesions on premolars or molars, were included in the study. Each patient underwent two microbiological investigations. First, the caries decay before treatment was assessed. Second, the dentine tissue was assessed after the removal of necrotic tooth decay and 24 s ozone application. The prevalence of oral streptococci was determined. In addition, we isolated Candida albicans, Enterococcus faecalis and Peptostreptococcus spp. from deep proximal caries lesion in caries-active patients. All microorganisms found in the deep proximal caries lesions were destroyed following 24 s ozone application. Our findings suggest that S. anginosus group, C. albicans and E. faecalis should be considered as an index for caries activity in caries-active patients, but additional studies are necessary to confirm this suggestion. This study demonstrated that 24 s of gaseous ozone application to the deep proximal caries lesions effectively eliminated microbial species.