Several studies have reported a complex microbial community in human breast milk. This community impacts the shape of the infant gut microbiota and consequently impacts host health. Lactobacillus is an important probiotic and has many applications in the functional food industry. This study isolated and evaluated the potential probiotic bacteria from human milk. Two Lactobacillus species, L. plantarum and L. pentosus, were isolated from the breast milk of Thai women. L. pentosus HM04-22, L. pentosus HM04-3, L. plantarum HM04-80, L. plantarum HM04-88 and L. plantarum HM01-1 showed good adhesion activity (> 55%) and resistance in gastric (pH 2) and bile (pH 8) conditions. Characterization of the probiotic properties indicated that all selected Lactobacillus isolates had anti-adhesion properties against Escherichia coli and Salmonella Typhimurium. Lactobacillus isolates protected Caco-2 cells from pathogen adhesion at 25-40%. In addition, the five selected strains presented anti-inflammatory properties by reducing interleukin (IL)-8 expression at 0.14 ± 0.16 to 0.52 ± 0.117-fold. However, the strains had no effect on the expression of tight junction genes, including zona occludens (ZO)-1, occludin and claudin-1. In conclusion, five selected Lactobacillus isolates from human milk were candidates for use as probiotics to promote health. However, more tests in animal models and clinical trials need to be performed.