<p>Breast milk harbors many bacteria that shape the newborn microbiota. The present study aimed to investigate the immunomodulatory properties of bacteria from selected Pakistani mother’s breast milk samples. The probiotic strains studied are associated with immune modulation in intestinal epithelial cells, showing potential probiotic properties to be used in a simulated human milk formula. Identification was carried out through 16&#xa0;S rRNA gene sequencing, which revealed the presence of 50% diversity of <i>Staphylococcus hominis</i> and 35% of <i>Staphylococcus epidermidis</i> among all the isolates. Probiotic bacteria are considered to protect the gastrointestinal tract (GIT) by adhering to the gut epithelial cells and reducing the population of pathogens. The cytotoxicity of <i>Staphylococcus hominis</i> MB614 and <i>Staphylococcus epidermidis</i> MB621 against epithelial cells was tested by co-culturing the strains with Caco-2 cells. Tested probiotic bacteria at concentrations, 1 × 10⁶, to 1 × 10² CFU/ml for 24&#xa0;h exhibited no noticeable cytotoxic effect on Caco-2 cells. <i>Staphylococcus hominis</i> MB614 and <i>Staphylococcus epidermidis</i> MB621 were examined for modulation of the immune system in human colon carcinoma cell lines (Caco-2) after the effect of the proinflammatory cytokine tumour necrosis factor α (TNF-α) by regulating the proinflammatory interleukin 8 (IL-8). <i>Staphylococcus hominis</i> MB614 and <i>Staphylococcus epidermidis</i> MB621 regulate TNF-α induced IL-8 production in a time-dependent manner. In conclusion, <i>Staphylococcus hominis</i> MB614 and <i>Staphylococcus epidermidis</i> MB621, isolated from human milk, when administered in sufficient amounts and for a certain duration, may be used to modulate inflammation inside the colon by up-regulating the production of IL-8, potentially enhancing innate immunity. This study with these probiotics suggested the modulation of the immune system and disease prevention in the human body.</p>