The purpose of this study was to evaluate the effects of the application of chitosan coating containing natamycin on the physicochemical and microbial properties of semi-hard cheese. Three cheese groups were prepared: samples without coating, samples coated with chitosan and with chitosan containing 0.50 mg mL−1 of natamycin, whose minimum inhibitory concentration was previously determinated on cheese surface. Microbiological analyses showed that natamycin coated samples presented a decrease on moulds/yeasts of 1.1 log (CFU g−1) compared to control after 27 days of storage. Addition of natamycin also affected O2 and CO2 permeability, increasing from 7.12 to 7.68 × 10−15 g·(Pa s m)−1, and from 10.69 to 64.58 × 10−14 g·(Pa s m)−1, respectively. The diffusion coefficient values of natamycin from the film to phosphate buffered saline solution and to the cheese were 3.60 × 10−10 and 1.29 × 10−12 cm2 s−1, respectively. This study demonstrated that chitosan-based coating/films can be used as release system containing natamycin to create an additional hurdle for moulds/yeasts in cheese thus contributing to extend its shelf-life.