Breast milk constitutes one of the most important sources of postnatal microbes. However, the influence of perinatal factors on the milk microbiome is still poorly understood. The aim of our study was to assess the impact of mode of delivery on the microbiome composition and diversity present in breast milk of healthy mothers. Mature milk samples (n=10) were taken from mothers after 1 month of exclusively breastfeeding. Microbiomes from milk samples were analyzed with 16S ribosomal RNA gene pyrosequencing and targeted quantitative polymerase chain reaction (PCR). Despite inter-individual variability in bacterial composition, The Principal Coordinates Analysis clearly separated milk microbiome from mothers with vaginal delivery (n=6) from those who undergo C-section (n=4). In addition, higher bacterial diversity and richness was found in milk samples from vaginal deliveries. Quantitative PCR data showed that higher levels of Bifidobacterium spp. were related significantly to lower levels of Staphylococcus spp. Despite the low sample size, our data suggest that mode of delivery has an important impact on milk microbiome composition. Further studies with larger sample sizes are needed to confirm these results and to understand the biological effects of C-section associated microbes on infant’s health.