To improve the mechanical properties and confer antimicrobial activity, transglutaminase (TGase) was used as a cross-linking agent and lysozyme (LYS) was incorporated as an antimicrobial agent to prepare novel active tilapia collagen (TC) films. While the difference in visual appearance was not obvious, the LYS incorporation increased the opacity of TC films. The water vapor permeability of all TGase cross-linked TC films was significantly (p < 0.05) lower than that of the control film (prepared without TGase and LYS). In addition, while the tensile strength and Young’s modulus of all TGase cross-linked TC films were significantly (p < 0.05) higher than those of the control film, elongation at break of all TGase cross-linked TC films was significantly (p < 0.05) lower than that of the control film. LYS incorporated TC films showed antimicrobial activity against E. coli, Staphylococcus aureus, Enterococcus faecium, Bacillus subtilis and Pseudomonas fluorescens. Collectively, TC films with improved physiochemical properties and antimicrobial activity have a good potential to serve as active food packaging materials.