BACKGROUND: Nasotracheal intubation can potentially result in microbial contamination from the upper respiratory tract to the lower respiratory tracts. However, an ideal nasotracheal disinfection method is yet to be determined. Therefore, we compared the disinfection effects between benzalkonium chloride and povidone iodine in nasotracheal intubation. METHODS: Overall, this study enrolled 53 patients aged 20-70 years who were classified into classes 1 and 2 as per American Society of Anesthesiologists-physical status and were scheduled to undergo general anesthesia with NTI. Patients who did not give consent (n = 2) and who has an allergy for BZK or PVI were excluded from the study. The patients were randomly divided into two groups on the basis of the disinfection method: BZK (n = 26, one patient was discontinued intervention) and PVI (n = 25). 50 patients were assessed finally. The subjects' nasal cavities were swabbed both before (A) and after disinfection (B), and the internal surface of the endotracheal tube was swabbed after extubation (C). The swabs were cultured on Brain heart infusion agar and Mannitol salt agar. The number of bacteria per swab was determined and the rates of change in bacterial count (B/A, C/B) were calculated. The growth inhibitory activity of the disinfectants on Staphylococcus aureus were also investigated in vitro. RESULTS: Although the initial disinfection effects (B/A) were inferior for benzalkonium chloride compared with those for povidone iodine, the effects were sustained for benzalkonium chloride (C/B). In the in vitro growth inhibitory assay against S. aureus, benzalkonium chloride showed higher inhibitory activity than povidone iodine. CONCLUSION: Although both disinfectants were inactivated or diffused/diluted over time, benzalkonium chloride maintained the threshold concentration and displayed antimicrobial effects longer than povidone iodine; therefore, benzalkonium chloride appeared to show a better sustained effect. Benzalkonium chloride can be used for creating a hygienic nasotracheal intubation environment with sustained sterilizing effects. TRIAL REGISTRATION: UMIN-CTR (Registration No. UMIN000029645 ). Registered 21 Oct 2017.