Infections caused by bacteria adhering to implant surfaces are one of the main reasons for the failure of the implants. In this study, polyurethane (PU), which is the most commonly used polymer in the production of medical devices, was synthesized and surfaces of polyurethane films were modified by chitosan (CH) grafting and heparin (Hep) immobilization in order to enhance anti-adhesiveness and antibacterial properties. Functional groups present on the surface, topographical shapes, and free energies of the polyurethane films were determined. Pristine polyurethane, chitosan-grafted polyurethane (PU-CH), and heparin immobilized polyurethane (PU-CH-Hep) films demonstrated high anti-adhesive efficacy against bacteria in the given order, where PU-CH-Hep was the most effective one. When PU-CH-Hep samples were incubated with different bacteria, complete death was observed for Pseudomonas aeruginosa (Gram negative), Staphylococcus aureus (Gram positive), and Staphylococcus epidermidis (Gram positive). Some living Escherichia coli (Gram negative) were observed after 24h of incubation. Pristine and modified polyurethane samples demonstrated no adverse effect on proliferation of L929 fibroblast cells and were found to be biocompatible according to MTT cytotoxicity tests.