The objective of the study was to evaluate the effect of hygiene measures in automatic milking units on the transmission of 3 mastitis pathogens considered to be mainly or partly transmitted from cow to cow during milking events. Two studies were conducted as within-herd experimental trials in 2 Danish commercial dairy herds (A and B) with automatic milking systems. Interventions to enhance hygiene were implemented on the automatic milking units. The 2 studies evaluated separate interventions. In herd A, the hygiene interventions were manual wash with the Lely foam unit and adjustments on the brush-mediated teat cleaning procedure. In herd B, the hygiene intervention included automatic disinfection spray on the upper surface of the brush motor and daily change of brushes. Composite milk samples were collected longitudinally at 3- or 4-wk intervals from all lactating cows. Additional milk samples were taken from cows entering or leaving the study groups. Milk samples were analyzed with quantitative PCR. A hidden Markov model implemented within a Bayesian framework was used to estimate the transmission probability. For analysis, 701 samples from 156 cows were used for herd A, and 1,349 samples from 390 cows were used for herd B. In the intervention group in herd B, transmission of Streptococcus agalactiae was reduced to 19% (95% posterior credibility interval: 0.00–64%) of the transmission in the control group, whereas transmission of Streptococcus dysgalactiae was reduced to 17% (95% posterior credibility interval: 0.00–85%) of transmission in the control group. This suggests that automatic spray on the upper surface of the brush motor with disinfectant along with daily change of brushes collectively reduced transmission of Strep. agalactiae and Strep. dysgalactiae. Results on Staphylococcus aureus in herd B and results on manual foam cleaning and brush-mediated teat cleaning adjustments in herd A were inconclusive.