IntroductionAnimal welfare is increasingly recognized as a key determinant of dairy herd health, yet its relationship with the presence of mastitis pathogens at the herd level remains insufficiently explored. This study aimed to evaluate the association between animal welfare indicators and the presence of major mastitis pathogens in bulk tank milk (BTM) from dairy cattle herds in the Aosta Valley, Italy, while also characterizing microbial profiles using bacteriological culture and polymerase chain reaction (PCR).MethodsA broad microbiological screening was performed on unique BTM samples from 490 individual farms. Concurrently, a subset of 355 small- and medium-scale dairy cattle farms underwent detailed animal welfare assessments using the standardized ClassyFarm protocol. Data were collected by experienced assessors in northern Italy in 2022, with each farm visited once.ResultsThe median lactating herd size was 33 cows (IQR: 57–18) and included Aosta Red Pied and Aosta Black Pied cattle breeds. All farms were tie-stall in the Aosta Valley, where herds are grazed outdoors for most of the year and kept indoors in tied-stalls during winter. BC identified at least one pathogen in 73.5% of samples, with most positive samples exhibiting low diversity, predominantly single (48.6%) or dual-species (41.1%) profiles. Staphylococcus aureus (N = 281) and Streptococcus agalactiae (N = 148) were the most frequently isolated pathogens. In contrast, PCR revealed prevalence of polymicrobial contamination, with the majority of samples harboring four or more bacterial species and 44.4% containing ≥5 species. Logistic regression models built using the binary PCR outcomes for the 355-farm subset demonstrated consistent negative associations between animal welfare indicators and the presence of both S. agalactiae and S. aureus. Higher total animal welfare scores were associated with reduced odds of detection (S. agalactiae: OR = 0.943, p ≤ 0.001; S. aureus: OR = 0.948, p = 0.005), as were animal-based measures, farm management, and housing and equipment scores.DiscussionThese findings provide evidence that improved animal welfare is associated with a lower likelihood of major mastitis pathogens in BTM.