Introduction: Poor agreements were previously described between tracheal wash (TW) and bronchoalveolar lavage fluid (BALF), as well as fungal detection by cytology and mycology culture. The link between moderate equine asthma (mEA) and detection of fungal elements in the airways remains controversial. Objectives: To determine the prevalence of fungal detection in TW and BALF and its association with diagnosis of mEA. Methods: Prospective study on 120 horses in active training or referred for respiratory disease. Horses were classified as “control” or “mEA” based on clinical examination, airway endoscopy and BALF. A sample was considered positive if at least one colony was identified by culture or at least one fungal element was observed on cytology. Results: Respectively, 35 and 85 horses were classified as “control” and “mEA”. No significant difference was observed between groups for fungal detection by cytology, regardless the sampling site. Prevalence of positive mycology culture was significantly higher for TW (89.4%) and BALF (31.8%) of mEA horses compared to controls (respectively 68,6% and 8.6%). Diagnosis of mEA was significantly associated with positive mycology culture on both TW (OR = 3.9) and BALF (OR = 5.0) Mycology culture on BALF exhibited high specificity (0.90) and high positive predictive value (0.91), unlike mycology culture on TW (respectively 0.76 and 0.31). Conclusion and clinical importance: Despite a significant association with asthma diagnosis, the high prevalence of fungal detection in TW of control horses precludes its clinical relevance. However, positive mycology culture on BALF represents a significant risk-factor of suffering mEA.