Klebsiella spp. is a relevant pathogen that can present acquired resistance to almost all available antibiotics, thus representing a serious threat for public health. While most studies have been focused on isolates causing community-acquired and nosocomial infections, little is known about the commensal isolates colonizing healthy subjects. We describe the molecular identification and the phenotypic characterization of commensal Klebsiella spp. from breast milk of healthy women and faeces from healthy breast-fed infants, which were compared with isolates from community-acquired infections and from a nosocomial NICU outbreak. The phylogenetic analysis of a 454-bp sequence of the rpoB gene was useful for species identification (K. pneumoniae, K. variicola, K. quasipneumoniae, K. oxytoca, K. grimontii, K. michiganensis, Raoultella planticola and R. ornithinolytica), previously misidentified as K. pneumoniae or K. oxytoca by biochemical methods. Globally, we report that commensal strains present virulence traits (virulence genes, siderophores and biofilms) comparable to community-acquired and NICU-infective isolates, thus suggesting that the human microbiota could constitute a reservoir for infection. Isolates causing NICU outbreak were multi-drug resistant (MDR) and ESBLs producers, although an imipenem-resistant commensal MDR K. quasipneumoniae isolate was also found. A commensal K. pneumoniae strain showed a potent bacteriocin-like inhibitory activity against MDR Klebsiella isolates, thus highlighting the potential role of commensal Klebsiella spp. in health and disease.