Potential triggers for equine asthma are allergens from hay and straw dusts, mold spores and storage mites. The contribution of these environmental trigger factors to equine asthma is still largely uncertain. The aim of this study was to compare results of four allergy tests from healthy and asthma-affected horses, and to evaluate the clinical relevance of allergens tested positive via specific inhalation provocation test. Fifteen horses were classified using a clinical scoring system as asthmatic (n = 9) or control (n = 6). Four different allergy tests (functional in vitro test, intradermal test, Fc-epsilon receptor test, and ELISA for allergen-specific IgE) were compared. A histamine inhalation provocation test as positive control was performed in all horses and the interpleural pressure was measured. In addition, two individual allergens were chosen for the allergen inhalation provocation test based on the results of the allergy tests and inhaled in increasing concentrations, until signs of dyspnea occurred. None of the four allergy tests could differentiate reliably between controls and asthma-affected horses. There was no agreement among the results of the four allergy tests. The interpleural pressure results showed a large individual variability. A clear positive reaction on the allergen inhalation provocation test was only detected in two asthma-affected horses 6 hours after allergen inhalation with Aspergillus fumigatus and Cladosporium herbarum. In most cases a purely type I immediate reaction is unlikely to be involved in causing the clinical signs of equine asthma. Because of a delayed reaction after allergen provocation in two horses, the involvement of cell-mediated type III or IV hypersensitivity may be possible. As all allergy tests used in this study can only detect IgE-mediated hypersensitivity, these tests are probably not suitable for an etiological diagnosis of equine asthma.