To evaluate the effect of the agitation of an ultrasonic photosensitizer during the antimicrobial photodynamic therapy (aPDT) in reducing multispecies biofilm composed of E. faecalis, C. albicans, and E. coli and endotoxins (LPS) within the root canals. Thirty lower premolars were contaminated and randomly divided into three groups (n = 10). Group 1 (G1) used saline solution as a control, G2 (aPDT) used a photosensitizer (0.005% methylene blue with 3 min) applied into the root canal followed by a red laser (100 mW power, 9 J of energy, for 90 s), and G3 (aPDT + U) used 0.005% methylene blue activated by ultrasound for 20 s followed by the application of a red laser as described in G2. The effectiveness of each protocol was determined through colony forming units per milliliter count (CFU/mL), and the levels of LPS were quantified by using the Limulus Amoebocyte Lysate technique. The data was statistically analyzed at a 5% level of significance. Viable bacteria were detected in all three groups, with a mean of 2.82 × 10 CFU/mL. The saline group was not effective in reducing viable counts or LPS levels (P > 0.05). The aPDT group resulted in a 56.27% reduction compared to baseline samples and aPDT + U promoted a 56.97% reduction in viable microorganisms from the biofilm. LPS was detected in all samples with a mean of 18.84 EU/mL. Saline was not effective in reducing LPS levels (P > 0.05). aPDT was effective in reducing LPS levels (P < 0.05) with a mean of 1.7 EU/mL. aPDT + U promoted additional LPS removal compared to the aPDT group (P < 0.05), with a mean of 0.02 EU/mL. For both CFU and LPS analyses, aPDT and aPDT + U were more effective than the control group, while aPDT + U was more effective than aPDT. In conclusion, ultrasonic activation of the photosensitizer during the aPDT increased antimicrobial activity against a mature multispecies biofilm, while lowering the LPS levels.