Alcohol-based disinfectants play an important role in the prevention of healthcare-acquired infection (HAI). We investigated whether pathogens present in mucus acquire resistance to alcohol-based disinfectants, and elucidated the underlying mechanism. Both the resistance of influenza A virus and Escherichia coli to alcohol-based disinfectants or ultraviolet irradiation and the diffusion rate of ethanol were determined in artificial mucus or sputum samples obtained from 27 individuals with acute upper respiratory infection. Pathogens in mucus (artificial mucus or sputum samples) were not completely inactivated by alcohol-based disinfectants (survival rate >10%), suggesting that the alcohol-based disinfectants were ineffective. Pathogen survival and mucus viscosity were strongly correlated (correlation coefficient >0.7, P < 0.001). Additionally, the ethanol diffusion rate decreased with increasing mucus viscosity, which contributed to ethanol resistance. Pronase treatment of sputum samples significantly decreased sputum viscosity and increased the disinfectant effect (P < 0.001 for all). In contrast, complete inactivation was achieved by ultraviolet irradiation independently of mucus viscosity. Thus, mucus viscosity contributes to resistance of pathogens to alcohol-based disinfectants by decreasing the alcohol diffusion rate. These findings can provide a basis for developing new strategies, including improved disinfectants, for overcoming HAI.