Aim To compare the incidence, intensity and prediction of postoperative pain after glide path preparation with manual, continuous rotary and a novel reciprocating glide path instrument. Methodology The study included 240 patients, who were treated by four specialists according to a planned treatment protocol. Following access cavity preparation and prior to glide path preparation, the subjects were randomly assigned to one of the three groups according to the glide path instrument by choosing a sealed envelope containing the group name: R-Pilot (VDW, Munich, Germany), ProGlider (Dentsply Sirona, Ballaigues, Switzerland) and stainless steel K-files (Dentsply Sirona; n = 80). Following glide path preparation, the teeth underwent standardized single visit root canal treatment procedures. The root canals were chemomechanically prepared using the ProTaper Next rotary system (Dentsply Sirona) under copious irrigation with 5.25% NaOCl. Final irrigation was performed with 17% EDTA and distilled water. Root fillings were placed using an epoxy resin sealer and gutta-percha with a cold lateral compaction technique. After coronal restorations were placed, the patients were discharged with a questionnaire about the incidence and intensity of pain at 6, 12, 18, 24, 48 and 72 h postoperatively. The data were analysed using chi-square, anova and Tukey tests and logistic regression analysis. Results The presence of preoperative pain (OR ranged between 3.5 and 14.3) and the glide path preparation techniques (OR between 2.2 and 4.1) were associated with significant effects on the incidence of postoperative pain when comparing manual versus engine-driven glide path preparation (P < 0.05). Patients in the R-Pilot (Mean VAS ranged between 1.57-0.21) and ProGlider (1.97-0.28) groups reported significantly less postoperative pain scores than those in the manual group (2.82-1.32; P < 0.05). There was no significant difference between R-Pilot and ProGlider groups regarding the postoperative pain scores (P > 0.05). Conclusion Preparation of glide paths with rotating or reciprocating NiTi instruments was associated with less postoperative pain levels and incidence compared to manual glide path preparation with no significant difference between rotating and reciprocating instruments. Preoperative pain was the most significant predictor for the occurrence of postoperative pain.