Objective To understand the influence of nursery care on the incidence of acute upper respiratory tract infection (AURI) and the change of pharyngeal pathogens in children, so as to provide reference for the development of effective AURI prevention measures and treatment strategies. Methods Ninety-one healthy preschoolers enrolled in a public kindergarten for the first time in this city were examined for pharyngeal swab pathogens before the first physical examination and after the first time of AURI in the kindergarten.And the influence of collective life on the change of pharyngeal pathogens was analyzed. Results There was no significant difference on the number of bacterial species isolated from pharynx swabs before and after AURI occurrence in 91 children, and the total proportion of bacteria (≥2+) after getting AURI was higher than the baseline (42.28% vs.33.77%, P=0.036).One case (1.1%) was positive for Mycoplasma, while chlamydia and 7 respiratory viruses were not detected before nursery care.However, 25 cases (27.47%) were positive for viruses and Mycoplasma after AURI, and the difference was statistically significant (χ²=37.111, P<0.001).Further analysis indicated that 51.65% were bacterial infections, 27.47% were non-bacterial infections, of which 5.50% were mixed infections.There was a tendency of concentrated distribution of common pathogens in the occurrence time of AURI. Conclusions There are mixed colonization of bacteria exists in the pharynx of healthy preschoolers, and the colonization of viruses, mycoplasma and Chlamydia is extremely rare.Collective life in kindergartens may induce the occurrence and transmission of AURI among children.So it is very important to strengthen child health care and disease prevention in kindergartens.