Disinfection and sterilization are needed for guaranteeing that medical and surgical instruments do not spread contagious microorganisms to patients. The aim of this study was to evaluate the efficacy of a simple manual technique of high-level disinfection (HLD) of flexible fiberoptic nasofibroscopes (FFNs) with wipes impregnated with a chlorine dioxide solution (Tristel Trio Wipes System—TTW) against a conventional automated washer machine (Soluscope ENT, Cimrex 12—AW). FFNs used in 62 patients undergoing endoscopy at an ENT clinic were sampled according to an aseptic procedure. For each nasoendoscopy, microbiological samples were taken at two times: (1) after a patient’s nasoendoscopy and (2) immediately after high-level disinfection. Ten microliters of each prepared sample were inoculated onto specific culture media for the detection of nasopharyngeal flora microorganisms. The microbiological results obtained from 62 post-disinfection samples revealed bacterial growth on two FFNs disinfected with AW, and five FFNs disinfected with TTW, but this difference is not statistically significant. None of the isolates were pathogenic bacteria. Our results are different than the results obtained by two previously published studies on the TTW system. In both studies, sampling was carried out by swabbing the tip and the handle surface of FFNs. This sampling method was the least effective method means of detecting bacteria on a surface. It can be concluded that the two disinfection systems allow providers to obtain a reduction of the saprophytic and pathogenic microbial load.