Hippobosca equina, a common ectoparasite of horses, poses a significant challenge to equine health worldwide. This study provides a molecular characterization of H. equina and examines its impact on host immune responses and oxidative stress. Genetic analysis of samples from various regions revealed notable homogeneity, indicating limited genetic variation among populations. Phylogenetic analysis demonstrated close genetic relationships among sequences from Egypt, France, Kazakhstan, and Portugal, while also showing comparable but more distant identities to sequences from China, Denmark, Finland, and other countries. Infested horses exhibited elevated levels of pro-inflammatory cytokines, including IL-6, TNF-α, and IFN-γ, relative to uninfested control horses, indicating a strong immune response to the parasite. Additionally, there was a significant increase in oxidative stress markers, including malondialdehyde, catalase, glutathione peroxidase, and nitric oxide, indicating substantial cellular damage. These findings highlight the dual impact of H. equina infestations on horse health, prompting both immune activation and oxidative stress. This study emphasizes the importance of targeted pest management strategies that consider the genetic uniformity of parasites and host physiological responses. By providing valuable insights into the complex host-parasite interactions between H. equina and equine hosts, this research enhances our understanding of equine ectoparasites and suggests potential avenues for improving horse health and welfare.