Understanding host–pathogen interactions in bovine mastitis is limited by the lack of physiologically relevant in vitro models, with most studies relying on immortalised bovine mammary epithelial cell lines. Organoids offer a three-dimensional, multicellular system that more closely recapitulates native tissue architecture and therefore represent a promising alternative for studying mammary gland infections. Here, we describe the development and optimisation of bovine mammary organoids (BMOs) as a platform for investigating host-pathogen interactions during mastitis infection.   Single cells were isolated from bovine mammary tissue, embedded in extracellular matrix domes, and cultured to generate BMOs. Early-passage (P0) cultures formed complex three-dimensional structures containing both luminal (KRT19⁺) and basal (KRT6⁺) epithelial cell populations, recapitulating key features of the bovine mammary gland in vivo. In contrast, later passages exhibited loss of luminal epithelial cells within these structures, resulting in reduced architectural complexity.   Together, these findings underpin ongoing work aimed at improving model robustness and reproducibility across passages through optimisation of the cell isolation strategies and the culture environment, including hydrogel-based scaffolds and alternative media. The optimised model is expected to support mastitis infection studies using well characterised bovine Streptococcus agalactiae and Staphylococcus aureus strains.