We wished to determine if Mycoplasma bovis infection can negatively impact milk quality and production in Holstein dairy cows. For this Research Communication, milk samples (271) from Holstein cows from 3 herds were screened for M. bovis by real-time PCR. Positive (n = 21) and negative animals (n = 21) were matched by herd, age, lactations and days in milk (DIM). Pairs were evaluated in 7 stages of lactation: D1–50, D51–100, D101–150, D151–200, D201–250, D251–300, and D ≥ 301. A mixed model was used to assess the effect of groups (M. bovis+ × M.bovis−), time (lactation) and groups × time interaction. Cows positive for M. bovis had lower average milk production per day and high somatic cells count (SCC).