Equine asthma is an inflammatory respiratory disorder, classified as mild-moderate (MEA) and severe (SEA). SEA is characterized by recurrent exacerbations, consisting of dyspnea, coughing and exercise intolerance; MEA causes poor performance, occasional cough and mucus hypersecretion. Although a precise pathogenesis is not completely understood, allergic mechanisms are considered an important pathophysiological feature of equine asthma. In equine medicine, intradermal testing (IDT) is effective in identifying hypersensitivity to specific allergens. However, to date, the studies about IDT in asthmatic horses obtained contradictory results. This study aims to evaluate IDT responses in MEA and SEA horses and to identify the most significant allergens. Thirty-eight asthmatic horses were enrolled and underwent IDT using 50 allergens; reactions were evaluated at 30 min, 4, 24 and 48 h and were assigned a score from 0 to 4. In SEA horses, the most frequent and strongest reactions were observed at 30 min and 4 h, suggesting the involvement of type I hypersensitivity; in MEA horses, also type IV hypersensitivity seemed to play a major role. Insects, and dog epithelium induced in MEA and SEA horses the most significant hypersensitivity responses and could therefore be considered as the main allergenic antigens in our geographic area.