Colonization of barley plants by the food-borne pathogens Salmonella enterica serovar typhimurium and three Listeria spp. (L. monocytogenes, L. ivanovii, L. innocua) was investigated in a monoxenic system. Herbaspirillum sp. N3 was used as a positive control and Escherichia coli HB101 as a negative control for endophytic root colonization. Colonization of the plants was tested 1–4 weeks after inoculation by determination of CFU, specific PCR assays and fluorescence in situ hybridization (FISH) with fluorescently labelled oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM). Both S. enterica strains were found as endophytic colonizers of barley roots and reached up to 2.3 × 106 CFU per g root fresh weight after surface sterilization. The three Listeria strains had 10-fold fewer cell numbers after surface sterilization on the roots and therefore were similar to the results of nonendophytic colonizers, such as E. coli HB101. The FISH/CSLM approach demonstrated not only high-density colonization of the root hairs and the root surface by S. enterica but also a spreading to subjacent rhizodermis layers and the inner root cortex. By contrast, the inoculated Listeria spp. colonized the root hair zone but did not colonize other parts of the root surface. Endophytic colonization of Listeria spp. was not observed. Finally, a systemic spreading of S. enterica to the plant shoot (stems and leaves) was demonstrated using a specific PCR analysis and plate count technique.