Human breast milk is of utmost importance in nurturing newborns as it furnishes them with vital nutrients and immune elements that are pivotal for their overall growth and maturation, majorly influencing the formation of infants’ gut microbiota. In situations where direct breastfeeding from the mother is unfeasible, pasteurized human donor breast milk sourced from human milk banks (HMBs) becomes an indispensable alternative for preterm and ill neonates. In this article, we delve into the bacterial makeup of pasteurized human milk found in HMBs. Our goals encompass exploring the range of microorganisms present, pinpointing potential sources of contamination throughout collection and processing, delving into the impact on healthcare, and evaluating the effectiveness of quality control steps. Recent breakthroughs in sequencing technologies have revealed an exciting discovery: Pasteurized human milk is teeming with a diverse array of bacteria. Among them are some friendly ones, such as bifidobacterium and lactobacillus, which have the potential to positively impact the gut health of newborns. While formerly believed to be sterile, human milk is today understood to be a dynamic ecosystem. The microbial diversity within pasteurized breast milk is influenced by maternal factors like diet and genetics. The risk of contamination arises during the collection, handling, and processing of milk. human milk banks follow strict quality control protocols, which mainly include donor screening, standardized collection, thorough pasteurization, and microbiological testing. The article raises questions about optimizing microbial diversity's benefits while mitigating risks, involving collaboration among clinicians, researchers, and HMBs. To ensure safety and quality, HMBs employ diverse strategies. Donor selection, sterile collection techniques, standardized pasteurization, and microbiological testing play critical roles. Proper storage and transportation practices are also essential. Staff training and quality assurance protocols further strengthen the safety and quality of pasteurized human milk, ultimately benefiting vulnerable neonates in need of nourishment and care.