UNLABELLED: The human milk (HM) microbiome is variable and depends on maternal, perinatal, and cultural-environmental factors. The diversity of the HM microbiome is crucial in the development of the child. The aim of the study was to assess the prevalence of bacteria (using culture-based methods) of Polish women with normal BMI, giving birth on time through vaginal delivery. METHODS: The research material consisted of human milk and swabs from the areola and nipple, before and after breastfeeding, derived from Polish women ( = 86). Classic culture methods were used to obtain multiple bacteria. Species identification of the grown colonies was performed using MALDI TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry). RESULTS: 120 species of bacteria were isolated, mainly from the genus and . Species specific only to human milk were identified (belonging to the following genera: , , , , , , , ) as well as species specific only to areola and nipple swabs after breastfeeding , , , ). It was confirmed that most species were present in all tested materials collected from one patient. CONCLUSIONS: The analysis carried out showed the presence of bacteria in the human milk of Polish women, including strains of lactic acid bacteria. The human milk microbiota may significantly influence the formation of the infant's intestinal microbiota, including some key genera, i.e., , , and , which were also isolated from the tested samples. The data presented here provide new data on culturable bacterial species isolated from breast milk from Polish women giving birth via vaginal delivery and potential routes of transmission from the neonate's oral cavity.