Infectious mastitis is a common condition among lactating women, with staphylococci and streptococci being the main aetiological agents. In this context, some lactobacilli strains isolated from breast milk appear to be particularly effective for treating mastitis and, therefore, constitute an attractive alternative to antibiotherapy. A (1)H NMR-based metabolomic approach was applied to detect metabolomic differences after consuming a probiotic strain (Lactobacillus salivarius PS2) in women with mastitis. 24h urine of women with lactational mastitis was collected at baseline and after 21 days of probiotic (PB) administration. Multivariate analysis (OSC-PLS-DA and hierarchical clustering) showed metabolome differences after PB treatment. The discriminant metabolites detected at baseline were lactose, and ibuprofen and acetaminophen (two pharmacological drugs commonly used for mastitis pain), while, after PB intake, creatine and the gut microbial co-metabolites hippurate and TMAO were detected. In addition, a voluntary desertion of the pharmacological drugs ibuprofen and acetaminophen was observed after probiotic administration. The application of NMR-based metabolomics enabled the identification of the overall effects of probiotic consumption among women suffering from mastitis and highlighted the potential of this approach in evaluating the outcomes of probiotics consumption. To our knowledge, this is the first time that this approach has been applied in women with mastitis during lactation.