Rosemary essential oil (REO) was used to develop an active film from chitosan. The effects of REO concentration (0.5, 1.0 and 1.5% v/v) on film properties were studied by measuring the physical, mechanical and optical properties of the REO-loaded films. Scanning electron microscopy and Fourier transform infrared (FTIR) spectroscopy were used to study microstructure and the interaction of the chitosan-based films. The solubility and water gain of the chitosan film decreased about 25% and 85%, respectively, by REO incorporation, up to 1.5% v/v, because of the interaction between hydrophilic groups of chitosan and REO as confirmed by FTIR. It was determined that REO improved the transparency of the films from 4.97 in neat chitosan up to 7.61; moreover, it reduced the films’ light transmission in UV light more than 25%. Films containing REO showed more antibacterial activity and total phenol content. The films containing REO showed potential to be used as active film in food preservation.