Background <p>Vaginitis is an inflammatory condition of the vagina commonly caused by infection with various pathogens. It is primarily characterized by vulvar or vaginal pruritus and increased vaginal discharge, which may exhibit abnormal color or odor. Causative agents include bacteria, fungi, and <i>Trichomonas vaginalis</i> (<i>T. vaginalis</i>), among others. Clinically, vaginitis may present as a monoinfection or as a mixed infection involving multiple pathogens. Notably, co-infections involving both <i>T. vaginalis</i> and fungi are relatively rare.</p> Case presentation <p>In our report, we described a case of vaginitis accompanied by <i>T. vaginalis</i> and fungal infection. Through microscopic examination of the patient’s vaginal discharge using the hanging-drop method, Wright-Giemsa staining method, and immunofluorescence staining method, numerous yeast cells, hyphae, and a small number of motile <i>T. vaginalis</i> were observed. To accurately identify the fungal strain, we conducted a pathogen culture. Colonies were detected growing on blood agar plates and Sabouraud agar plates. Mass spectrometry analysis was then employed to identify the isolated strain as <i>Candida albicans</i> (<i>C. albicans</i>). Based on these findings, clinicians formulated a targeted treatment plan.</p> Conclusions <p>Vaginitis with a co-infection of <i>T. vaginalis</i> and fungi is relatively uncommon, and it is easy to overlook during laboratory examinations. Multiple detection methods should be employed to verify test results, providing robust support for clinical diagnosis and treatment. This case highlights the importance of comprehensive microbiological investigation in patients with atypical presentations.</p>