Despite enormous efforts, complete animal tuberculosis (TB) eradication has only been achieved in few regions. Epidemiological analyses are key to identify TB risk factors and set up targeted biosecurity measures. Here, we conducted an in-depth characterization of 84 extensive beef cattle farms from a high TB prevalence region in Western Spain, and assessed how farm management and wildlife presence on farms contribute to cattle TB risk. Twenty-six out of 84 variables were associated with cattle farm TB positivity. Farm management variables associated with TB positivity included older cattle, larger herd size, highly fragmented farm structure and greater connectivity between farms. TB-positive farms provided supplemental feed over a higher number of months, used calf feeders, and had higher number of waterholes. Detecting Eurasian wild boar (Sus scrofa), red fox (Vulpes vulpes), European badger (Meles meles), roe deer (Capreolus capreolus), or Egyptian mongoose (Herpestes ichneumon) on cattle farms was also associated with farm TB positivity. The best ordinal regression model indicated that in farms with a large herd size (> 167 animals) the odds of being positive or recurrently positive (versus negative) was 7.34 (95% CI = 2.43–23.51) times higher that of farms with small herd size. Further, for every additional host species detected in the farm premises, the odds of being TB-positive increased 56%. We conclude that both cattle management and wildlife need to be targeted for successful TB control in grazing-based farming systems.