The main aim of this work is to develop a transparent bioactive cellulose acetate (CA) film for food packaging applications. The aim of the study is also to optimize the production methods for development of bulk and surface immobilized bioactive CA films. The vacuum drying method was optimized for the production of transparent CA films. The direct infusion and UV-assisted surface immobilization methods were investigated for the development of bioactive CA films. Reduction in the crystalline nature of CA was observed to be responsible for the production of transparent CA films. Thymol, with high antioxidant and antimicrobial properties, was examined to be the major active compound (>40%) present in the polar fraction of oregano. Retention of active compounds analyzed by High Performance Liquid Chromatography (HPLC), surface morphology analyzed by Atomic Force Microscopy (AFM), and surface chemistry analyzed by X-ray Photoelectron Spectroscopy (XPS) proved the efficiency of UV-assisted surface immobilization method. Acetyl cellulose films treated with UV irradiation at 312 nm were examined to have higher retention of active compounds. The bioactive CA films produced by bulk and surface immobilization methods showed >90% and ∼65% thymol retention, respectively. The UV-assisted surface immobilization method was found to decrease the mechanical and barrier properties of CA film. The bioactive CA films produced by bulk and surface immobilization methods were found to have retained the antioxidant and antimicrobial properties of the thymol.