This randomized clinical trial evaluated the effectiveness of supplemental photodynamic therapy (PDT) in optimizing the removal of bacteria and endotoxins from primarily infected root canals after one-visit and two-visit treatments.Twenty-four primarily infected root canals with apical periodontitis were selected and randomly divided into one-visit (n=12) and two-visit treatments (n=12). Chemo-mechanical preparation (CMP) was performed by using the single-file reciprocating technique + 2.5% NaOCL and a final rinse with 17% Ethylenediamine tetraacetic acid (EDTA). The photosensitizer agent (methylene blue 10 mg/mL) was applied to root canals for 60 seconds and submitted to laser with a potency of 60 milliwatts (mW) and energy density of 129 J/cm2 for 120 seconds after CMP in the one-visit treatment and after 14-day inter-appointment medication with Ca(OH)2 + saline solution (SSL) in the two-visit treatment. Samples were collected before and after root canal procedures. Endotoxins were quantified by chromogenic limulus amebocyte lysate assay. Culture techniques were used to determine bacterial colony-forming unit counts.