In food emulsions such as raw milk, the lipid droplets exhibit an interfacial layer possibly the site of interactions with bacteria, including the shiga toxin-producing Escherichia coli (STEC). Some pathogenic E. coli strains can adhere to intestinal cells after ingestion or to ligands that could modulate their pathogenicity such as those present at the surface of emulsion lipid droplets. The objectives of this study were to investigate the preferential localization of E. coli cells, AEEC 4315-A strain belonging to the O26:H11 serotype, as a function of the surface composition of emulsion lipid droplets i.e. milk fat globule membrane (MFGM) surrounding raw milk fat globules, milk polar lipids or whole milk proteins. The emulsions were artificially spiked with E. coli from an overnight culture in Brain Heart Infusion broth. Then, the E. coli cells were enumerated in the lipid droplet enriched creams and in the skimmed phases recovered after centrifugation. The localization of the E. coli cells in the creams was observed by confocal laser scanning microscopy (CLSM). This study revealed the role played by the surface properties of dairy lipid droplets on the adhesion of E. coli. For milk fat globules, the concentrations of E. coli in the creams were about 10 times higher than in the aqueous phase. In the processed emulsions, E. coli cells were concentrated in the aqueous phase. The CLSM images showed the adhesion of E. coli to the MFGM containing glycoproteins and their mobility as a function of time in the aqueous phase surrounding lipid droplets. This study provided additional scientific information supporting a specific affinity of E. coli for the MFGM of raw milk fat globules.