Different adjunct methods have been proposed to reduce microorganisms during endodontic treatment. This study aimed to evaluate microbial reduction in vitro in the root canals of extracted primary teeth contaminated with Enterococcus faecalis (ATCC), following antimicrobial photodynamic therapy (aPDT). Twenty-one root canals of primary teeth, decals from the repository of a research group were contaminated and divided into three groups (7 roots per group): the control group (CG) underwent chemical-mechanical preparation (CMP) with mechanized instrumentation and smear layer removal; intervention group 1 (G1) underwent aPDT without CMP; and intervention group 2 (G2) underwent CMP followed by aPDT. Significant bacterial reductions were observed across all the groups (p = 0.001), with final mean bacterial loads statistically lower than the initial mean levels (p < 0.05, Mann-Whitney test). The G2 group demonstrated the highest mean microbial reduction, with 5 roots achieving complete bacterial elimination (100% reduction). Comparatively, the mean colony-forming unit (CFU) reduction values in G2 were comparable to those in CG (p = 0.318), and both were significantly higher than in G1 (p = 0.001 for CG vs. G1; p = 0.001 for G2 vs. G1). Under the conditions tested, aPDT served as an effective adjunct technique to pulpectomy, but did not outperform conventional pulpectomy performed alone.