Background: Fungi are ubiquitous in horses' environment. Their contribution to the pathophysiology of severe asthma (SA) is acknowledged, while controversies remain for mild–moderate asthma (MA). Objectives: We hypothesised that fungi were a risk factor for asthma. Our objective was to compare different combinations of analytical methods (cytology, culture) and sampling sites (tracheal wash (TW), bronchoalveolar lavage fluid (BALF)) in relation to clinical status (control, MA, SA). Study design: Prospective cross-sectional study. Methods: The study population included asymptomatic racing horses in the field and horses referred to the hospital for respiratory investigations. Fungi were detected by cytology and identified by mycology on TW and pooled BALF. Chi-square tests were used for prevalence comparison between groups and association with clinical investigations. Results: A total of 155 horses (85 MA, 35 SA and 35 controls) were included in the study. The overall proportions of fungal detection in TW ranged from 45.7% to 89.4% among groups. The prevalence of fungal detection in BALF was significantly lower by cytology for SA (5.7%) than MA horses (23.6%) and significantly higher by culture for MA horses (31.8%) than controls (8.6%). Fungal detection by culture in BALF was significantly associated with high tracheal mucus score, high neutrophil proportions in BALF and diagnosis of MA. Main limitations: Mycology was only performed in pooled BALF and environment was not sampled. Conclusion and clinical importance: Fungi were significantly more prevalent in the airways of MA horses than SA and/or controls. Fungal detection in TW, either by cytology or culture, was uninformative in a clinical context. Fungal detection by culture (but not cytology) in BALF represents a risk factor for MA. Ethical animal research: Approved by the regional Ethic Committee for Clinical and Epidemiological Veterinary Research (CERVO-2020-3-V). Informed consent: Owners gave consent for their animals' inclusion. Competing interests: None declared. Funding: IFCE: Grant numbers