BACKGROUND: Fungal esophagitis (FE) is caused by fungal invasion of the esophageal mucosa. Under endoscopy, the esophageal mucosa shows edema, congestion, erosion, and ulceration, and bleeds easily when touched, and the surface of the mucosa is covered with small white spots like "bean curd residue". Clinical cases showing typical FE under endoscopic imaging but negative esophageal mucosal brush (referred to as suspected FE) have increased the difficulty and challenge of clinical diagnosis and treatment. At present, the esophageal fungal flora of suspected case has not been thoroughly studied. AIM: To characterize the fungal flora in FE, suspected FE, and the esophageal normal controls (NCs), and to identify marker species to improve detection of FE. METHODS: This was a case-control study. A total of 19 patients with FE, 16 with suspected FE, and 10 NCs were selected by endoscopy. The esophageal cell brush samples of each group were sequenced by internal transcribed spacer (ITS) 1 and analyzed by bioinformatics. RESULTS: In FE and suspected FE patients, species richness, species diversity and species evenness, as measured by the Chao1 index, Shannon index and Pielou index, were lower than in the NCs, and the comparison between the FE and NCs was the most significant ( CONCLUSION: ITS showed that composition of the fungal community was similar in the FE and suspected FE groups. ITS can be used as an auxiliary diagnostic method for FE and provide a theoretical basis for follow-up diagnosis and treatment.