Unsedated peroral endoscopy, including ultrathin endoscopy (UE) and conventional endoscopy (CE), is feasible in clinical practice but requires improved endoscopic operability and patient tolerance. Currently, the impact of the breathing method on these factors remains unclear. We conducted the first randomized controlled trial comparing oral breathing (OB) and nasal breathing (NB) during both UE and CE to assess their influence. About 252 eligible patients undergoing CE or UE were randomly assigned to OB or NB groups. Endoscopists and patients rated endoscopic operability and patient tolerance using a 100-mm visual analog scale. Visibility from the oral cavity to the middle pharynx was recorded. OB led to a higher rate of improved visibility from the oral cavity to the middle pharynx compared with NB, ranging from 79.3% to 81.0%. Multivariate correlation analyses showed significantly lower visual analog scale scores for endoscopic operability with OB compared with NB in both UE and CE groups ( P < 0.05). No significant differences were found in the overall evaluation of patient tolerance between OB and NB groups in UE and CE, whereas the smaller diameter of UE exhibited better patient tolerance compared with CE. Discriminant analysis comparing endoscope types and breathing methods revealed that UE with OB outperformed other combinations in the overall evaluation of endoscopic operability and patient tolerance ( P < 0.05). OB facilitates endoscopic operability compared with NB in peroral endoscopy. UE with OB is recommended as the preferred choice for unsedated peroral endoscopy in daily practice.