Controlling Listeria in food is a major challenge, especially because it can persist for years in food processing plants. The best option to control this pathogen is the implementation of effective cleaning and disinfection procedures that guarantee the safety and quality of the final products. In addition, consumer trends are changing, being more aware of the importance of food safety and demanding natural foods, minimally processed and free of chemical additives. For this reason, the current consumption model is focusing on the development of preservatives of natural origin, from plants or microorganisms. In sum, this study aimed to evaluate the antimicrobial effectiveness of a citrus extract formulation rich in flavonoids against several L. monocytogenes and L. innocua strains, using in vitro test (agar diffusion test, minimum bactericidal concentration (MBC), and time-kill curves) and challenge test in food trials (carne mechada, salami, fresh salmon, lettuce, brine, and mozzarella cheese). The results presented in this work show that citrus extract, at doses of 5 and 10%, had a relevant antimicrobial activity in vitro against the target strains tested. Besides this, citrus extract applied on the surface of food had a significant antilisterial activity, mainly in carne mechada and mozzarella cheese, with reductions of up to eight logarithmic units with respect to the control. These results suggest that citrus extract can be considered a promising tool to improve the hygienic quality of ready-to-eat foods.