In laying hens, Escherichia coli (E. coli) and Gallibacterium anatis (G. anatis) are considered the two major pathogens causing reproductive tract disorders, either as single infections or as co-infections. Vertical transmission has been confirmed for E. coli but remains to be clearly demonstrated for G. anatis. The aim of the present study was to investigate the ability of both G. anatis and E. coli at eggshell transmission using an embryonated egg dipping model, and to investigate the possible interaction between the two organisms in an embryonated egg injection model. Embryonated eggs were dipped into brain heart infusion broth containing 10⁸ CFU/ml either of G. anatis 12656-12 liver, E. coli ST95 or E. coli ST141, respectively. E. coli ST95 and ST141 were re-isolated from the interior egg contents in 60% (12/20) and 85% (17/20) of the eggs, respectively, while G. anatis 12656-12 was only re-isolated from the interior egg contents in 6.7% (3/45) eggs. Eggs were injected with 10–1000 CFU of either G. anatis 12656-12, E. coli ST95 or ST141 into the allantoic cavity. As few as 10 CFU of G. anatis 12656-12 resulted in 100% mortality within 24 h post injection whereas the E. coli injected embryos all died at 48 h post injection. Significant difference in CFU counts were observed for G. anatis when compared G. anatis injection group with either of the two G. anatis – E. coli co-injection groups. Sixteen hours post injection, a significant difference in embryo mortality could be observed when comparing co-injected embryonated eggs (G. anatis and E. coli) and single-injected (G. anatis or E. coli) embryonated eggs. In conclusion, bacterial transmission via the eggshell was demonstrated for both G. anatis and E. coli although at different magnitudes. The embryonated egg injection model revealed that G. anatis in particular was highly pathogenic when exposed directly to the developing embryo.