The European polecat (Mustela putorius) almost became extinct in Britain in the early twentieth century, but populations are now recovering. As seen in other endangered carnivore populations, disease is one potential threat to recovery. This study assessed exposure of wild polecats (n = 149) to three, multi-host pathogens which could limit reproduction and/or cause morbidity and mortality. Serum, lung and brain samples were collected from polecats which died from 2011 to 2016 across Britain. Exposure to Toxoplasma gondii and 12 Leptospira serovars was assessed serologically by antibody detection using the latex agglutination test and microscopic agglutination test, respectively, and the presence of canine distemper virus (CDV) RNA in lung and brain tissue samples was assessed using PCR. Generalised linear models were used to test for relationships between exposure to each pathogen and season, sex, age, and location. All organ samples tested PCR negative for CDV (95% CI 0.00–0.05%). There was evidence of frequent exposure to T. gondii with a recorded seroprevalence of 71.8% (95% CI 64.2–79.4%) and moderate exposure to Leptospira serovars, 14.5% (95% CI 8.6–20.4%). Season, sex, age, and location were not significantly associated with exposure to T. gondii or Leptospira serovars. Evidence of exposure to T. gondii and Leptospira serovars in European polecats could potentially affect mortality, longevity or fecundity. Further studies are warranted to assess the impact of these pathogens on polecat populations in Britain.