Besides the advances in endodontics, there are situations in which surgery is necessary to retain a tooth that otherwise would be extracted. This study analyzes the microbial reduction after conventional periapical surgery followed by antimicrobial photodynamic therapy (aPDT) in 3 years follows up. Twenty-eight teeth needing periapical surgery were enrolled in this study. Microbiological samples were taken after: (1) accessing the lesion, (2) surgical procedure, and (3) aPDT. The teeth received a full mucoperiosteal flap, osteotomy with a high-speed bur, manual curettage of lesion and of the root, root-end resection and retrograde cavities prepared using ultrasonic retro-tips. After the conventional procedure the cavities received an aqueous solution of methylene blue (60 μM, 3 min) and were irradiated with a diode laser λ=660 nm (6 min, 15 J). After aPDT, a retrograde filling with mineral trioxide aggregate, flap re-positioning, and sutures conventionally ended the procedure. In all the cases, a periapical X-ray exam was taken before and after the surgical procedure. The microbiological samples showed an overall significant reduction, surgical procedure achieved a mean reduction of about 3.5 log while aPDT achieved a 5 log reduction. After 36 months of follow up the patients had, in average a reduction of 78% of the periapical lesion area. It appears that surgical endodontic treatment associated with antimicrobial photodynamic therapy highly improves the microbial reduction compared to the traditional technique and this could directly affect the treatment prognosis and periapical lesion healing.