The utilization of biopolymers incorporated with antimicrobial agents is extremely interesting in the development of environmentally friendly functional materials for food packaging and other applications. In this study, the effect of calcium oxide (CaO) on the morphological, mechanical, thermal, and hydrophilic properties as well as the antimicrobial activity of carboxymethyl chitosan (CMCH) bio-composite films was investigated. The CMCH was synthesized from shrimp chitosan through carboxymethylation, whereas the CaO was synthesized via a co-precipitation method with polyethylene glycol as a stabilizer. The CMCH-CaO bio-composite films were prepared by the addition of synthesized CaO into the synthesized CMCH using a facile solution casting method. As confirmed by XRD and SEM, the synthesized CaO has a cubic shape, with an average crystalline size of 25.84 nm. The synthesized CaO exhibited excellent antimicrobial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (>99.9% R). The addition of CaO into CMCH improved the mechanical and hydrophobic properties of the CMCH-CaO films. However, it resulted in a slight decrease in thermal stability. Notably, the CMCH-CaO10% films exhibited exceptional antimicrobial activity against E. coli (98.8% R) and S. aureus (91.8% R). As a result, such bio-composite films can be applied as an active packaging material for fruit, vegetable, or meat products.