The non-specific adsorption of proteins to surfaces in contact with biofluids constitutes a major problem in the biomedical and biotechnological field, due to the initiation of biofilm formation and the resulting improper function of devices. Therefore, non-fouling surfaces modified with poly(ethylene glycol) (PEG) are usually applied. In this study, we report the synthesis of triethoxysilane modified glycerol based polymers of linear and branched architecture for the preparation of covalently attached monolayers on glass. Evaluation of the biocompatibility of these surfaces was performed in comparison to bare non-coated glass, hydrophobic hexadecane modified glass, and mPEG modified glass as the controls. Protein adsorption of BSA and fibrinogen (1 mg · mL−1 in PBS) after 4 and 24 h immersion was reduced by more than 96 and 90%, respectively, compared to the adsorption on bare glass substrates. In addition, mouse NIH-3T3 fibroblast cells showed only marginal adhesion on the polyglycerol and mPEG coated slides after 3 and 7 d incubation in cell suspension, which demonstrates the long-term stability of the applied glass coatings. The non-adhesive properties of these coatings were further reflected in bacterial adhesion tests of Escherichia coli K12 and three clinically relevant Gram-positive and negative strains (Staphylococcus aureus, Pseudomonas aeruginosa, and Aeromonas hydrophila), since linear polyglycerol (LPG(OH)), linear poly(methyl glycerol) (LPG(OMe)), and hyperbranched polyglycerol (HPG) reduced the adhesion for all tested strains by more than 99% compared to bare glass. Therefore, polyglycerol derivatives present an excellent non-fouling surface coating as an alternative to PEG with feasibility for surface modification of various substrates.