Background: Subclinical mastitis (SCM) is an inflammatory condition of the mammary gland. We examined the effects of SCM on human milk (HM) composition, infant growth, and HM intake in a mother–infant cohort from seven European countries. Methods: HM samples were obtained from 305 mothers at 2, 17, 30, 60, 90, and 120 days postpartum. SCM status was assessed using HM Sodium (Na): Potassium (K) ratio >0.6. Levels of different macro- and micronutrients were analyzed in HM. Results: SCM prevalence in the first month of lactation was 35.4%. Mean gestational age at delivery was lower and birth by C-section higher in SCM mothers (p ≤ 0.001). HM concentrations of lactose, DHA, linolenic acid, calcium, and phosphorous (p < 0.05 for all) was lower, while total protein, alpha-lactalbumin, lactoferrin, albumin, arachidonic acid to DHA ratio, n-6 to n-3 ratio and minerals (iron, selenium, manganese, zinc, and copper) were higher (p < 0.001 for all) in mothers with SCM. There were no differences in infant growth and HM intake between non-SCM and SCM groups. Conclusion: We document, for the first time, in a large European standardized and longitudinal study, a high prevalence of SCM in early lactation and demonstrate that SCM is associated with significant changes in the macro- and micronutrient composition of HM. Future studies exploring the relation of SCM with breastfeeding behaviors and developmental outcomes are warranted.