In dry climates, livestock farming contributes to waterhole creation and maintenance, thereby contributing to biodiversity conservation. However, these lentic water bodies also represent a critical environmental connection between microorganisms and their vertebrate hosts since the water can facilitate pathogen persistence and transmission. Therefore, interventions for tuberculosis (TB) risk mitigation at the wildlife-livestock interface often focus on segregating host species at water points. We hypothesized that waterhole characteristics modulate their use by vertebrates and subsequent pathogen exposure risk. We visited 298 waterholes on 80 TB-positive and 40 TB-negative cattle farms in Spain to assess differences in waterhole characteristics and identify possible management implications. There was an average of 2.7 waterholes per farm. This represents 0.02 waterholes per km² of farmland and 3.5 m² of lentic waterbodies per km² of farmland. Among the studied waterholes, 95% were man-made. Waterholes on TB-positive farms were 42% closer to covering vegetation than waterholes located on TB-negative ones. Farms with man-made waterholes showed a higher risk of TB than those with natural ones. The density of waterholes per surface unit was negatively associated with the farm TB risk. Waterholes placed on TB-positive farms were more intensively trampled by livestock. The best model explained farm positivity to TB as a function of the distance from the waterholes to the nearest cover vegetation, the aquatic vegetation richness found in the waterhole, the intensity of livestock use (trampling), the surrounding waterhole density, and the interaction between waterhole perimeter and the total number of signs of potential wildlife TB hosts per waterhole. Identifying the key waterhole features related to infection risk might allow designing One Health–inspired biosecurity measures such as increasing the number of waterholes, placing new waterholes farther away from cover, or fencing-out cattle from wildlife-rich waterholes to balance biodiversity conservation and animal health needs in extensive grazing systems.