Objective To understand the distribution characteristics of fungi on object surface in hospital environment, and provide reference for the scientific and precise formulation of environment control strategies based on fungal in clinic. Methods From December 7 to 23, 2023, a total of 60 environmental specimens of 19 categories in 6 departments of a large tertiary first-class hospital were collected and divided into water-related environmental specimen group, complete facade environmental specimen group, and sanitary ware environmental specimen group. 18S rRNA sequencing was performed on specimens with fungi detected. Results Fungal detection rate of environmental specimenswas 20.00% (12/60). Sink in the department of endocrinology had the highest fungal colony count (15 CFU/cm²), followed by the air outlet of air disinfection device in the department of thoracic surgery and the internal part of a faucet in the department of endocrinology (both 10 CFU/cm²). The water-related environmental specimen group detected most diverse fungal genera (14 species), with high relative abundances of Aspergillus (100%), Meyerozyma (99.06%), Ophiocordyceps genus (95.63%), and Kodamaea (87.86%). The air outlet of air disinfection device was detected with a high abundance of Chaetomium (44.08%) and Corollospora (39.71%). There was no statistically significant difference in the α-diversity (Shannon and Simpson indices, P values of 0.661 and 0.568, respectively) and β-diversity (P=0.712) among the three environmental specimens. Conclusion Under the routine implementation of basic environmental cleaning and disinfection in medical institutions, fungi are in a low prevalence in the environment. However, moist surfaces and air disinfection device are prone to fungal colonization, and it is necessary to strengthen daily monitoring and take corresponding intervention measures to reduce the risk of infection.