Salmonella enterica ssp. enterica serovars Enteritidis (SE) and Gallinarum (SG) cause different diseases in chickens. However, both are able to reach the blood stream where heterophils and monocytes are potentially able to phagocytose and kill the pathogens. Using an ex vivo chicken whole blood infection model, we compared the complex interactions of the differentially host-adapted SE and SG with immune cells in blood samples of two White Leghorn chicken lines showing different laying performance (WLA: high producer; R11: low producer). In order to examine the dynamic interaction between peripheral blood leucocytes and the Salmonella serovars, we performed flow cytometric analyses and survival assays measuring (i) leucocyte numbers, (ii) pathogen association with immune cells, (iii) Salmonella viability and (iv) immune gene transcription in infected whole blood over a four-hour co-culture period. Inoculation of blood from the two chicken lines with Salmonella led primarily to an interaction of the bacteria with monocytes, followed by heterophils and thrombocytes. We found higher proportions of monocytes associated with SE than with SG. In blood samples of high producing chickens, a decrease in the numbers of both heterophils and Salmonella was observed. The Salmonella challenge induced transcription of interleukin-8 (IL-8) which was more pronounced in SG- than SE-inoculated blood of R11. In conclusion, the stronger interaction of monocytes with SE than SG and the better survivability of Salmonella in blood of low-producer chickens shows that the host-pathogen interaction and the strength of the immune defence depend on both the Salmonella serovar and the chicken line.