This study integrates proteome analysis of human breast milk (HBM) from a homogeneous group of mothers who are of similar age and live in the same geographical area, along with an analysis of essential and potentially toxic elements in HBM in relation to lifestyle and environmental factors. This preliminary proteomic study, which examined 11 samples of HBM from lactating women, identified a total of 1619 proteins across all samples, revealing significant differences in proteomes influenced by lactation stages, parity, and exposure to tobacco smoke. The pilot study aimed to explore the feasibility of correlating certain proteins with several elements, considered as indicators of tobacco smoke and environmental influences on HBM. Notably, a clear and significant correlation was found between altered calcium content in HBM and the proteome fraction associated with calcium-binding proteins. The findings suggest that all analyzed factors impact the HBM proteome and the activity of certain enzymes.