Breastfeeding is recommended for the first 6 months of life. Many bioactive compounds of the human milk (HM) support the development of the intestinal immune system and barrier functions in infants. Our hypothesis was that HM microbiota contributes to these health benefits. Our objective was to characterise in vitro the role of HM bacteria, either individually or combined in synthetic communities (SynCom), on gut homeostasis. A collection of bacterial isolates, reflecting HM microbiota composition, was made from 28 healthy mothers exclusively breastfeeding. Firstly, the immunomodulatory profile of 84 HM bacterial isolates belonging to 38 species was characterized using blood mononuclear cells (PBMC). Secondly, the impact of a subset of 29 strains was deeply investigated on epithelial immune and barrier functions using a quadricellular (Caco2, HT29-MTX-E12, M cell, THP1 cells) model of the intestinal epithelium. Strains were characterized for their ability to modulate cellular IL-10 and TNF-α production and the expression of genes related to the barrier, immune and apoptosis/proliferation functions. Based on these results, 2 SynComs were designed and characterized on the quadricellular model. HM bacteria displayed a large range of immunomodulatory properties. Using MultiDimensionate Scaling (MDS) on IL-10 and TNF-α production by PBMC, isolates were classified into 5 groups with specific signatures, highlighting the anti- and/or pro-inflammatory profiles of HM bacteria. Further, the MDS analysis of cytokine production and gene expressions of the quadricellular model stimulated by each of the 29 bacteria, classified strains into 3 groups named Quadri1, 2 and 3, according to their immunomodulatory activity and their impact on the epithelial barrier function. The composition of each group did not reveal major taxonomic biases between the 3 groups, but a diversity of the HM bacteria impact on gut epithelium within each genus or species. Quadri3 and, to a lesser extent, Quadri1 strains stimulated the immune function whereas Quadri2 hardly affected it. Besides, Quadri1 and 3 strains reinforced the epithelial barrier whereas an opposite effect was observed with Quadri2 strains. Finally, strains belonging to prevalent HM genera and with contrasted immunomodulatory profiles were assembled in two HM-like SynCom of 11 strains. The 2 Syncoms displayed different immunomodulatory properties, yet less contrasted than individual strains, whereas they both exhibited beneficial impact on barrier function. This study showed the great diversity of immunomodulatory potential and impact on the barrier function of HM bacteria, individually or assembled in SynCom, highlighting the potential of the HM microbiota to modulate the intestinal development.