Surface modification with hydrophilic polymers is used extensively in a range of biomedical applications in order to control the interactions with proteins, cells and bacteria. However, the architecture of the polymer layers play an important role in the ability to either totally prevent non-specific interactions or engage with proteins to enhance cell responses to biomaterials. In this study we utilized a range of poly(ethylene glycol)-thiol derivatives to functionalise surface plasmon resonance (SPR) gold sensor chips, and studied their influence upon protein adsorption. Specifically, we aimed to change the polymer graft density and functionality in order to capture proteins and preserve their activity in cell culture, but at the same time find an optimal surface that also prevented bacterial adhesion. The PEG-thiol graft density was varied by changing the polymer concentration and the results showed that under specific conditions, the PEG-thiol density was highest when grafted at the highest concentration for all derivatives. The modified surfaces were characterised with X-ray photoelectron spectroscopy (XPS), SPR and water contact angle measurements. Additionally, all modified surfaces were exposed to fibronectin solutions, cell and bacterial attachment assays were performed with mesenchymal stem cells (MSCs) and Pseudomonas aeruginosa, respectively. We found that the lowest fibronectin adsorption was observed on surfaces having the highest PEG-thiol graft density, P. aeruginosa attachment was very low on all surfaces, and it was observed that MSC attachment gradually increased as the PEG-thiol density decreased, reaching the maximum levels at medium and low PEG-thiol surface coverages.