Human milk (HM) is the best feeding option for preterm infants; however, when mother’s own milk (MOM) is not available, pasteurized donor human milk (DHM) is the best alternative. In this study, we profiled DHM microbiota (19 samples) using 16S rRNA amplicon sequencing and compared its compositional features with the MOM microbiota (14 samples) from mothers who delivered prematurely (PT-MOM). As a secondary study aim, we assessed the specific effect of pasteurization on the characteristics of the DHM microbiota. DHM showed significantly higher alpha diversity and significant segregation from PT-MOM. Compositionally, the PT-MOM microbiota had a significantly higher proportion of Staphylococcus than DHM, with Streptococcus tending to be and Pseudomonas being significantly overrepresented in DHM compared with the PT-MOM samples. Furthermore, pasteurization affected the HM microbiota structure, with a trend towards greater biodiversity and some compositional differences following pasteurization. This pilot study provided further evidence on the HM microbial ecosystem, demonstrating that the DHM microbiota differs from the PT-MOM microbiota, possibly due to inherent differences between HM donors and mothers delivering prematurely, and that pasteurization per se impacts the HM microbiota. Knowledge about HM microbiota needs to be acquired by investigating the effect of DHM processing to develop strategies aimed at improving DHM quality while guaranteeing its microbiological safety.