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To achieve an effective hygiene management within equine clinical environments, an objective assessment of given hygiene structures, work flows and patient-associated risk factors is imperative together with a continuing surveillance of multi-drug resistant (MDR) pathogens frequently associated with nosocomial infections. In the first part of this interventional study, critical hygiene areas and procedures were identified and assessed with respect to their distinct risks for both, the equine patients and the veterinary personnel during an observation period of six months. In addition, a continuing surveillance of site infections (SI) associated with MDR pathogens was implemented for two important distinct medical indications (colic surgery, open injuries). Then, the intervention process started with practical hygiene education of the personnel including a strong focus on hand hygiene compliance. A bundle of further actions was implemented, including standard operation procedures (SOPs) for bandage change, wound- and catheter management and application of drugs. Horse owners were informed about correct behavior in the stables, regular room cleaning plans were accomplished and an appropriate mandatory hygiene instruction for students, residents and guests was introduced. An increase of 190% was documented for consumption of alcoholic hand disinfection in the second study phase, while the overall rate of recorded SI in colic- and injury patients decreased by 12.1 and 6.6%, respectively. Noted cases of MDR pathogens in SI decreased by 9.5%. Here we provide strong evidence for the necessity of continuing surveillance of defined site infections and the beneficial effects of a targeted hygiene management implemented and evaluated in a large equine clinic, given that all structural measures and responsible authorities act in concert.