Biofilms are aggregates of microorganisms that adhere to the surface of certain substances and produce extracellular polymers to improve their resistance to stress. However, only some Bifidobacterium strains can form biofilms on abiotic surfaces. The study evaluated the biofilm-forming abilities of six Bifidobacterium strains on grape seed flour (GSF) with a particle size of 80–120 μm. Bifidobacterium animalis, Bifidobacterium bifidum, and Bifidobacterium adolescentis formed weak biofilms, whereas Bifidobacterium pseudo, Bifibacterium breve, and Bifidobacterium longum formed strong biofilms on GSF. B. pseudo cells formed biofilm particles with a diameter of 2 mm, and their cell numbers reached 2.04 × 109 CFU/g at 32 h. There were almost no viable cells in the culture without GSF after 60 h; however, the number of biofilm cells on GSF was still over 106 CFU/g, indicating that biofilms improve cell survival. Field-emission scanning electron microscopy revealed the following stages in the bifidobacterial biofilm formation process: adsorption to GSF, secretion of extracellular substance, biofilm maturation and dispersal, and cell death. Collectively, these results strongly suggest that GSF is beneficial for Bifidobacterial biofilm growth, providing a theoretical basis for the production of highly resistant probiotics in the food industry.