Abstract Background Studies in people have found neutrophil gelatinase‐associated lipocalin (NGAL) concentrations are increased in asthma and can be used to distinguish between asthma subtypes. NGAL has not yet been investigated in equine asthma (EA). Objectives To investigate the ability of NGAL concentrations in bronchoalveolar lavage (BAL) fluid and serum to distinguish between control horses, horses with mild–moderate EA (MEA) and horses with severe EA (SEA). Study design Retrospective cross‐sectional study. Methods Details of endoscopic examination including tracheal mucus score (TMS, scale 0–5) and BAL cytology performed on 227 horses were extracted from records and NGAL concentrations were measured on stored serum and BAL fluid samples. The horses were divided into groups (control group n = 73, MEA n = 98, SEA n = 56) based on clinical signs and BAL cytology results. Differences between groups were evaluated with the Mann–Whitney test and correlation between BAL NGAL, serum NGAL, and BAL cytology were evaluated using Spearman's correlation. Results BAL NGAL concentrations were higher in EA than in control horses (median: 25.6 and 13.3 μg/L, respectively, p < 0.001). Concentrations of NGAL in BAL differed between groups, with higher concentrations in MEA than in control horses (median: 18.5 and 13.3 μg/L, respectively, p < 0.001), and higher concentrations in SEA than in MEA horses (median: 54.1 and 18.5 μg/L, respectively, p < 0.001). BAL NGAL concentration differed between horses with TMS ≤2 an >2 (median 15.6 and 21.1 μg/L, respectively, p = 0.004). No differences were found in serum NGAL concentration between any of the groups. Main limitation Only 66 of the 227 (29%) horses had haematology and serum NGAL measured. Conclusion BAL NGAL concentration differed between control and EA and reflected severity of disease. These results justify further research into the potential of NGAL as a biomarker of EA.