Prevention of microbial contamination of surfaces is one of the biggest challenges for biomedical applications. Establishing a stable, easily produced, highly antibacterial surface coating offers an efficient solution but remains a technical difficulty. Here, we report on a new approach to create an in situ hydrogel film-coating on glass surfaces made by enzymatic cross-linking under physiological conditions. The cross-linking is catalyzed by horseradish peroxidase (HRP)/glucose oxidase (GOD)-coupled cascade reactions in the presence of glucose and results in 3D dendritic polyglycerol (dPG) scaffolds bound to the surface of glass. These scaffolds continuously release H 2O 2 as long as glucose is present in the system. The resultant polymeric coating is highly stable, bacterial-repellent, and functions under physiological conditions. Challenged with high loads of bacteria (OD 540 = 1.0), this novel hydrogel and glucose-amended coating reduced the cell viability of Pseudomonas putida (Gram-negative) by 100% and Staphylococcus aureus (Gram-positive) by ≥40%, respectively. Moreover, glucose-stimulated production of H 2O 2 by the coating system was sufficient to kill both test bacteria (at low titers) with >99.99% efficiency within 24 h. In the presence of glucose, this platform produces a coating with high effectiveness against bacterial adhesion and survival that can be envisioned for the applications in the glucose-associated medical/oral devices.