Background: The use of an untargeted metabolomic approach to investigate biofluids of respiratory origin is of increasing interest in human and veterinary lung research. Considering the high incidence of equine asthma (> 14%) within horse population and the importance of this animal model for human disease, we aimed to investigate the metabolomic profile of bronchoalveolar lavage fluid (BALF) and exhaled breath condensate (EBC) in healthy and asthmatic horses. Results: On the basis of clinical, endoscopic and BALF cytology findings, 6 horses with severe asthma (Group A) and 6 healthy horses (Group C) were included in the study. 1 H-NMR analysis was used to identified metabolites in BALF and EBC samples. Metabolomic analysis allowed to identify and quantify 12 metabolites in BALF and seven metabolites in EBC. Among respiratory metabolites, myo-inositol, formate, glycerol and isopropanol in BALF, and methanol and ethanol in EBC, differed between groups (p < 0.05). Conclusions: The application of metabolomic studies to investigate equine asthma using minimally invasive diagnostic methods, such as EBC metabolomics, provided promising results. According to our research, the study of selective profiles of BALF and EBC metabolites might be useful for identifying molecules like myo-inositol and methanol as possible biomarkers for airways diseases in horses.