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Background Ultraconservative removal of carious tissue is becoming increasingly highlighted for management of deep caries lesions, and combined with an antimicrobial photochemistry-based treatment modality (PACT), this approach can be enhanced favoring dental tissue repair and preservation. The aim was to investigate the effectiveness of PACT using a light emitting diode (LED) associated with a photosensitizer toluidine blue ortho (TBO) on deep caries lesions. Methods For that, a single blind, randomized, controlled, split-mouth clinical trial where 45 patients with at least two deep carious lesions on permanent posterior teeth was performed. The primary intervention was deep caries lesion management with disinfection of remaining dentin tissue using PACT. Bacterial counts were measured following treatments as the main outcome. The remaining dentinal samples of each lesion were treated with either non-PACT-control or PACT. The PACT procedure were characterized by 100 μg mL−1 TBO followed by 94 J cm−2 LED irradiation. Samples of dentin were collected before and immediately after treatments for microbiological analysis of total viable bacteria, mutans streptococci and Lactobacillus spp. counts. Microbial reduction was data were submitted to unpaired t test (α = 5%). Results PACT led to statistically significant reductions in mutans streptococci (1.08 ± 1.20 log), Lactobacillus spp. (1.69 ± 1.37 log), and total viable bacteria (1.07 ± 1.01 log) compared to the control, which showed log reductions respectively of 0.05 ± 0.49, 0.52 ± 0.89, and 0.47 ± 0.77 for the same microorganisms. Conclusion Dentin from deep carious lesions treated with PACT showed a decrease in cariogenic microbial load.