Equine asthma (EA) is the most prevalent chronic lung disease in horses. The immunological processes are only partially understood. Neutrophils are the primary effector cell in severe EA (sEA) and essential components of the innate immune defence. One defence mechanism is the release of neutrophil extracellular traps (NETs). NETs have the ability to capture and kill pathogens; however, they can also contribute to autoimmune responses, chronic inflammation and host damage if not properly regulated. The aim of this study was to characterise the role of NETs in the pathogenesis of EA. Analysis of BALF revealed the highest numbers of NET activated cells in sEA, in addition to elevated levels of equine cathelicidin and reduced DNase activity. Isolated blood neutrophils from horses with EA showed increased NET formation in vitro, which correlated with the clinical severity and a decrease in the cellular cholesterol content. Neutrophil cholesterol further correlated with clinical parameters associated with disease severity. The presence of circulating anti-neutrophil cytoplasmic antibodies (ANCAs) suggested local and systemic NET-related alterations. The findings of this study provide novel insights into immunological alterations in EA, with potential for the development of diagnostic and therapeutic strategies targeting NET formation and associated immune dysfunction.