Abstract Nowadays a sequence of tests for evaluation of sterilized biomaterial includes an initial set of tests in vitro, both biological (cell culture) and non-biological (mechanical tests). In this paper the cytotoxicity of a sterilized polyurethane film, in order to use as biomaterial, has been investigated. For this purpose NCO-terminated urethane prepolymer in medical quality was synthesized without ingredients beside monomers (polyethylene glycol/castor oil and toluene diisocyanate). The cured prepolymer films were prepared under ambient conditions due to the reaction of free NCO-groups of prepolymer with air moisture. The polyurethane films were sterilized by gamma-ray (25 kGy). The surface structure of sterilized polyurethane film was observed by SEM and compared to that of the unsterilized film. Also, the in vitro interaction of fibroblast cells and sterilized polyurethane film in culture medium containing serum was evaluated in comparison with control samples. Results showed no signs of cell toxicity.