This study aimed to improve the physical, mechanical, and biological properties of a monolayer pectin (P) film containing nanoemulsifiedtrans-Cinnamaldehyde (TC) by incorporating it between inner and outer layers of ethylcellulose (EC). The nanoemulsion had an average size of 103.93 nm and a zeta potential of −46 mV. The addition of the nanoemulsion increased the opacity of the film, reduced its moisture absorption capacity, and improved its antimicrobial activity. However, the tensile strength and elongation at break of the pectin films decreased after the incorporation of nanoemulsions. Multilayer films (EC/P/EC) showed a higher resistance to breaking and better extensibility compared to monolayer films. The antimicrobial activity of both mono and multilayer films was effective in inhibiting the growth of foodborne bacteria during storage of ground beef patties at 8 °C for 10 days. This study suggests that biodegradable antimicrobial multilayer packaging films can be effectively designed and applied in the food packaging industry.