Maternal milk is an important source of essential nutrients for the optimal growth of infants. Breastfeeding provides a continuous supply of beneficial bacteria to colonize the infant gastrointestinal tract (GIT) and offers health benefits for disease prevention and immunity. The purpose of this study was to isolate novel probiotic strains from the breast milk of native Pakistani mothers and to evaluate their probiotic potential. We isolated 21 strains of bacteria from the colostrum and mature milk of 20 healthy mothers, who had vaginal deliveries and were not taking antibiotics. After phenotypic and genotypic characterization, these isolates were tested for survival in the GIT using in vitro acid and bile tests. Nine strains showing good acid tolerance were assessed for their growth rate, bile resistance and ability to hydrolyze bile salts. Out of the four Lactobacillus isolates adjudged to be most promising as probiotics, three were Lactobacillus fermentum strains and one was a strain of Lactobacillus oris . This study demonstrates that human milk is a viable source of commensal bacteria beneficial to both adults and babies.