Decontamination of bacterial biofilms formed on food-contact surfaces is a serious challenge in the food industry. In this study, a food-grade antimicrobial system was developed using yeast-based microcarriers and essential oil (EOs; thymol) to inactivate bacterial biofilms on food-contact surfaces. A bio-inspired method that involves pre-treatment of yeast microcarriers with simulated intestinal fluid (SIF) was employed to modulate the release of the encapsulated EOs. The effectiveness of the developed antimicrobial delivery system was evaluated based on physicochemical characterization, antimicrobial activities against planktonic bacterial cells in the presence of organic matter, affinity to bind bacterial biofilms, and co-incubation and residual antimicrobial activities against bacterial biofilms. The results illustrate that pre-treatment of yeast-based microcarriers with SIF significantly (p < 0.05) enhanced the release of the encapsulated EOs without influencing the encapsulation yield and binding of yeast-based microcarriers with bacterial biofilms. Strong antibiofilm activities were exhibited by the thymol encapsulated in the pre-digested yeast cells (pdYC@Thymol) compared to free thymol or thymol encapsulated in the undigested yeast microcarriers. Populations of Listeria innocua in biofilms (ca. 7.54 log CFU/cm2) decreased to ca. 2.63 log CFU/cm2 after 1 h of co-incubation with the pdYC@Thymol suspensions and further decreased to ca. 0.85 log CFU/cm2 after removal of the loosely bound pdYC@Thymol particles. Overall, this study suggests that the combined effects of the targeted delivery of the EOs via encapsulation in yeast-based microcarriers and facilitated release of the encapsulated EOs upon binding with bacterial biofilms can effectively inactivate the bacterial biofilms formed on food-contact surfaces, thereby enhancing the microbiological safety of the food-related environments.