Zwitterionic silica coatings for surface functionalization are greatly prominent because of their simple and fast preparation, high availability, and effective antifouling properties. In this work, two zwitterionic sulfobetaine silane coatings, i.e., mono-SBSi and tris-SBSi, were deposited on glass surfaces and tested for antifouling of biological material and biofilm using human cancer cell and seawater, respectively. The used zwitterionic precursors mono-SBSi and tris-SBSi differ by the number of hydrolyzable silane groups: mono-SBSi contains one trimethoxysilane group, whereas tris-SBSi contains three of these functions. First, X-ray photoelectron spectroscopy indicates the successful grafting of zwitterionic coatings onto a glass surface. Characterization using atomic force microscopy shows the different morphologies and roughness of the two coatings. The glass surface became more hydrophilic after the grafting of zwitterionic coatings than the bare glass substrate. The antifouling properties of two coatings were evaluated via human cancer cell adsorption. Interestingly, the tris-SBSi coating displays a significantly lower level of cell adsorption compared to that of both mono-SBSi coating and the non-modified control surface. The same trend was observed for biofilm formation in seawater. Finally, the toxicity of mono-SBSi and tris-SBSi coatings was evaluated on zebrafish embryos, indicating the good biocompatibility of both coatings. Our results indicate interesting antifouling properties of zwitterionic coatings. The chemical constitution of the used precursor has an impact on the antifouling properties of the formed coating: the tris-SBSi-based zwitterionic silica coatings display improved antifouling properties compared to those of the mono-SBSi-based coating. Besides, the use of trisilylated precursors should result in the formation of more resistant and robust coatings due to the higher number of grafting functions. For all these reasons, we anticipate that tris-SBSi coatings will open new perspectives for antifouling applications for biological environments and implants.