Breast-milk is an important source of bacteria for the colonization of the infant‘s gut. The aim of our study was to isolate and identify bacteria from samples of breast-milk of 32 women and from fecal samples of their breast-fed infants. Antimicrobial activity of isolates was also performed. A total of 155 isolates were characterized by phenotypic tests and identified by 16S rDNA sequencing analysis. The isolates belonged to 6 different species of acid lactic bacteria (LAB) and Staphylococcus epidermidis. Enterococcus faecium was the most frequently isolated species (40.8%) and faeces (42.5%). According to the mother’s lifestyle, we noticed that the genus Enterococcus was the most frequently isolated from rural mother’s milk as well as urban mother’s milk. Lactobacillus fermentum (P < 0.05) and Staphylococcus epidermidis(P < 0.01) were isolated only from rural mother’s milk. An antimicrobial activity was observed in 30 strains from 148 LAB, the higher level of antagonist was with E.faecium (35 mm). The observed results showed that the isolated strains from rural mother’s milk were different from that urban mother’s milk. Eventual studies can be carried out about lifestyle and nutrition of mothers to explain the effect on the flora found in the milk and feces infants.