Intestinal organoids are self-organized three dimensional (3D) structures composed of a layer of polarized intestinal epithelial cells surrounding a hollow lumen. They recapitulate in vitro the intestinal multicelular composition, architecture and physiology. The aim of this work was to set up organoid models for studying zoonotic pathogens such as Salmonella and Toxoplasma gondii. T. gondii’s sexual cycle is restricted to felid’s intestines, which are characterized by an excess of linoleic acid given by the lack of delta-6-desaturase activity. “Felinized” murine intestinal organoids were generated for triggering T. gondii’s sexual differentiation in vitro. For this purpose, murine intestinal organoids from C57BL/6 mice were established from crypt isolated intestinal stem cells (2D or 3D) and incubated in the presence of 20 μM delta-6-desaturase inhibitor and 200 μM linoleic acid. Under these conditions no cytotoxicity of felinizing compounds was observed until 5 days of incubation. To optimize T. gondii’s infection, intestinal organoids were incubated with tachyzoites (at three distinct multiplicities of infection, MOIs) and evaluated by immunofluorescence assays (IFAs) at three time points post-infection. In order to set up a Salmonella infection model, intestinal organoids from farm animals (cow and sheep) were established and characterized by light microscopy and RT-PCR of specific markers. Forward steps will involve bovine intestinal organoids exposure to Salmonella enterica reporter strains at different MOIs, and bacteria invasion/proliferation evaluation at two time points after infection by extra and intracellular bacteria quantification and IFAs. Our results highlight the versatile uses of intestinal organoids as a powerful in vitro tool for modeling zoonotic diseases, contributing to the principle of reducing the use of experimental animal models.