Abstract Objectives Detecting bacterial activity is considered a promising approach to monitor shifts from symbiosis to dysbiosis in oral microbiome. The present study aimed at investigating both the relative bacterial activity and the lactate dehydrogenase (ldh) gene expression of caries-associated bacteria in a site-specific natural biofilm. Material and methods Sixty subjects (age, mean ± SE: 30.1 ± 1.4) were allocated to two groups: caries-free subjects (CF) or caries-active subjects (CA). CF presented one sound surface (CFS, n = 30). CA presented two donor sites: a cavitated caries lesion (CAC, n = 30) and a sound reference surface (CAS, n = 30). Real-time quantitative PCR (q-PCR) on species or genus level and total bacteria was performed targeting the 16S gene, the 16S rRNA, the ldh gene, and the ldh mRNA (increasing 16S ribosomal RNA copy numbers can function as an indicator of increased energy metabolism). As the 16S rRNA abundance represents the number of ribosomes, while the 16S gene abundance represents the number of genomes, the quotient of the relative abundances functions as a measure for the relative bacterial activity (%). Results Both lactobacilli and S. mutans showed the highest relative bacterial activity in CAC ((mean ± SE) 218 ± 60% and 61 ± 16%, respectively) and the lowest values for both sound reference surfaces (69 ± 48%; 8 ± 3%). Significant differences were found between CAC and CAS as well as between CAC and CFS for both lactobacilli and S. mutans (p < 0.05). The ldh gene expression of lactobacilli and S. mutans only showed moderate values in CAC (1.90E+03 ± 2.11E+03; 2.08E+04 ± 4.44E+04 transcripts/μl) and CFS (2.04E+03 ± 2.74E+03; 8.16E+03 ± 6.64E+03 transcripts/μl); consequently no significant differences were detected. Conclusion and clinical relevance Caries-associated bacteria (lactobacilli and S. mutans) showed the highest relative bacterial activity in plaque of cavitated lesions, the lowest in sound surfaces, allowing the detection of a significant activity shift in health and disease for caries-active patients. However, no significant differences in ldh gene expression could be determined.