Human milk (HM) microbiota is increasingly studied for its potential health benefits. However, the physiological state of HM bacteria and consequently their effects on gut homeostasis remain a question. This study investigated the physiological state of the HM microbiota by characterizing its cultivable fraction as it might be at the point of ingestion and assessing the effects of digestion, in the specific context of the immature infant digestive tract, on the cultivability and immunomodulatory properties of six HM strains representative of prevalent genera in HM. Twenty-eight HM samples were analysed by 16 S metabarcoding either directly on raw milk (raw milk microbiota, RM) or on the complete cultivable fraction obtained from seven non-selective media (cultivable milk microbiota, CM). This approach enabled a more in-depth investigation of CM than conventional methods based on the individual sequencing of a subset of isolates and resulted in a moderate gain in diversity within each HM sample. It confirmed that diversity was lower in CM than in RM, with ~ 7 versus 69 genera per sample in CM and RM respectively, and an under-representation of strictly anaerobic genera in CM. In vitro infant gastrointestinal digestion resulted in overall good survival of the 6 HM strains but partial or complete loss of their immunomodulatory properties on the monocyte THP1 cell line, except for a Staphylococcus epidermidis strain that gained immunomodulatory potential. These results highlight the potential of HM bacteria to survive during the infant gastrointestinal digestion and interact with the intestinal epithelium and immune system, as well as the importance of considering the digestion process when evaluating host-bacteria interactions.