Background: Protein carbonyl levels are the most frequently used biomarker of protein oxidation in several human diseases, including cancer. Breast cancer, a worldwide disease with increasing incidence, develops from ductal/lobular epithelium from which nipple aspirate fluid can be collected and analysed to assess tissue metabolic activity. Our aims were to perform an exploratory investigation on the protein carbonyl accumulation in breast secretions from healthy and cancer patients and its correlation with lipid peroxidation markers. Methods: Protein carbonyls were determined by ELISA in 288 Nipple Aspirate Fluids (NAF) from Control, Pre-malignant and Cancer patients. Results: Significantly higher protein carbonyl concentration was found in NAF from breast cancer (BC) patients compared to Control subjects. Cancer patients accumulated in NAF significantly higher levels of carbonyls in post-menopausal condition. A significant inverse relationship between carbonyls and 8-F2α-isoprostanes in NAF was found in Cancer patients. NAF levels of protein carbonyls are significantly higher in women with pre-malignant conditions than in healthy subjects. Conclusions: Our results support the hypothesis that oxidative stress in breast microenvironment plays a role in breast cancer; measurement of protein and lipid oxidative products in NAF may improve the identification of women at increased breast cancer risk.