Objective To explore the clinical infection distribution and antifungal resistance characteristics of the Stephanoascus ciferrii (S. ciferrii) complex. Methods Clinical fungal culture and isolation data from Nanjing Drum Tower Hospital from 2012 to 2023 were retrospectively analyzed. The annual isolation quantity, proportion, and distribution of infection sites of S. ciferrii were statistically analyzed. The minimum inhibitory concentrations (MICs) of isolated strains against common antifungal agents were detected by ATB Fungus 3 fungal susceptibility test kit, and their resistance was analyzed. Results From 2012 to 2023, a total of 26 602 fungal strains were isolated in this hospital, including 79 strains of the S. ciferrii complex. Compared with 2012-2017, the isolation rate of S. ciferrii complex increased from 2018 to 2023 (0.06% vs 0.43%, P<0.001). There were 69 cases of superficial infection, including infection of ear canal (n=67) and skin (n=2). 92.75% (n=64) of the patients were immunocompetent individuals. There were 10 cases of invasive infection, including infection of urinary tract (n=5), respiratory tract (n=2), biliary tract (n=1), pacemaker pouch (n=1), and vagina (n=1). 70.00% of the patients had other underlying diseases, including 3 cases of immunosuppressed patients who died due to combined infection with the S. ciferrii complex. The MICs detection results showed that the MIC₅₀ and MIC₉₀ of fluconazole were 32 μg/mL and 128 μg/mL, respectively; the MIC₅₀ and MIC₉₀ of amphotericin B were 0.5 μg/mL and 2 μg/mL, respectively. Conclusion S. ciferrii complex mainly causes ear canal infection and has the dual pathogenic ability to cause superficial and invasive infection. The isolated strains showed higher MIC values for fluconazole and lower MIC values for amphotericin B. The mortality is high in immunosuppressed patients combined with invasive infection caused by S. ciferrii.