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Background Human milk microbiota plays a role in the bacterial colonization of the neonatal gut, which has important consequences in the health and development of the newborn. However, there are few studies about the vertical transfer of bacteria from mother to infant in Latin American populations. Methods We performed a cross-sectional study characterizing the bacterial diversity of 67 human milk-neonatal stool pairs by high-throughput sequencing of V3-16S rDNA libraries, to assess the effect of the human milk microbiota on the bacterial composition of the neonate’s gut at early days. Results Human milk showed higher microbial diversity as compared to the neonatal stool. Members of the Staphylococcaceae and Sphingomonadaceae families were more prevalent in human milk, whereas the Pseudomonadaceae family, Clostridium and Bifidobacterium genera were in the neonatal stool. The delivery mode showed association with the neonatal gut microbiota diversity, but not with the human milk microbiota diversity; for instance, neonates born by C-section showed greater richness and diversity in stool microbiota than those born vaginally. We found 25 bacterial taxa shared by both ecosystems and 67.7% of bacteria found in neonate stool were predicted to originate from human milk. This study contributes to the knowledge of human milk and neonatal stool microbiota in healthy Mexican population and supports the idea of vertical mother-neonate transmission through exclusive breastfeeding.