Simple SummaryEquine periodontal disease (EPD) is a painful syndrome in horses that causes significant health problems. Despite its clinical importance, very few investigations on its etiopathogenesis have been performed. This study investigates the association of different bacterial species, including the red complex bacteria (RCB, i.e.,Treponema denticola,Tannerella forsythiaandPorphyromonas gingivalis),Fusobacterium nucleatum,Veilonella parvula, andPrevotella intermedia, with periodontal disease and other risk factors.Tannerellaspp. andTreponemaspp. were detected with a significantly higher proportion in horses affected by EPD than in healthy animals. Overall, the investigated pathogens, belonging to RCB, were detected in a low number of horses. Age was found to be the main risk factor, with horses aged older than 20 years being at higher risk of EPD. A very high rate of coinfection was statistically associated with EPD, and, thus, EPD supported the multiagent etiology of this equine complex syndrome.AbstractEquine periodontal disease (EPD) is a painful oral inflammatory syndrome characterized by multifactorial pathogenesis. Although it is well known that bacterial proliferation and consequent gingivitis are caused by the decomposition process of food residues, in hypsodont species, the pathogenetic role and the different bacterial species involved in the progression of EPD must be fully clarified. This study aimed to investigate the association of bacteria, including the complex red bacteria (RCB), with EPD, and to evaluate possible EPD risk factors. Bacterial species, includingTreponema denticola,Tannerella forsythia,Porphyromonas gingivalis(belonging to the RCB),Fusobacterium nucleatum,Veilonella parvula, andPrevotella intermedia, were investigated in 125 oral swabs from healthy and EPD-affected horses using real-time multiplex PCR. Subsequently, possible risk factors (i.e., age, gender, and breed of the animals and type of feed used) were evaluated using univariate and multivariate analyses.Tannerellaspp. andTreponemaspp. were detected in a significantly higher proportion of horses affected by EPD than in healthy animals, although pathogens belonging to RCB were detected in low number of horses. At the same time, none of the investigated pathogens was significantly associated with a particular stage of disease severity. Horses aged older than 20 years were at higher risk of EPD. The high rate of coinfection, statistically associated with EPD, supports the hypothesis that EPD is a complex syndrome characterized by the possible simultaneous involvement of several pathogens and an increased risk depending on the animal’s age. Constant oral hygiene is the best prevention to prevent and treat the disease, especially in old animals.