Background To meet the nutritional needs of preterm infants, multicomponent nutrient fortifiers are added to human milk. The fortified human milk (FHM) product changes the physical and biochemical characteristics of the milk. We questioned whether such physical-chemical changes in the milk would alter intrinsic probiotic bacterial activity. The objective of the study was to evaluate the effect of osmolality and pH on the growth of probiotic bacterial species intrinsic to human milk. Methods Human milk samples (n = 26) were collected from mothers in the neonatal intensive care unit (NICU) and stored at -20°C until analyzed. The samples were thawed and divided into three portions. Human milk fortifiers (HMFs) were added to two portions to prepare concentrations of FHM. The remaining portion was the unfortified control sample. Each sample was then divided into two parts. One part (baseline) was used to measure the osmolality and pH and plated on selective agar to enumerate the growth of lactobacilli and bifidobacteria species. The remaining part was incubated at 37°C for 24 h to further test bacterial integrity (post-incubation) and then the same measurements were made (osmolality, pH, bacterial colony counts). Results When compared with unfortified milk at baseline, osmolality increased and pH decreased significantly after the addition of HMFs. Lactobacilli and bifidobacteria colony counts did not differ among the groups pre-incubation. Post-incubation lactobacilli and bifidobacteria increased in all the groups. Conclusion The appropriate addition of HMFs differentially affected the osmolality and pH of the milk. These physical changes did not affect the growth of probiotic bacterial species.