Aflatoxin B1 (AFB1) is a mycotoxin considered a potent carcinogen for humans that contaminates a wide range of crops. Various strategies have been established to reduce or block the synthesis of AFB1 in food and feed. The use of aqueous extracts derived from plants with high antioxidant activity has been a subject of study in recent years due to their efficacy in inhibiting AFB1. In this study, we assessed the effect of Aloysia citrodora aqueous extract on Aspergillus flavus growth and on AFB1 production. A bio-guided fractionation followed by High Performance Liquid Chromatography (HPLC) and Mass spectrometry analysis of the active fraction were applied to identify the candidate molecules responsible for the dose-effect inhibition of AFB1 synthesis. Our results revealed that polyphenols are the molecules implicated in AFB1 inhibition, achieving almost a total inhibition of the toxin production (99%). We identified luteolin-7-diglucuronide as one of the main constituents in A. citrodora extract, and demonstrated that it is able to inhibit, by itself, AFB1 production by 57%. This is the first study demonstrating the anti-Aflatoxin B1 effect of this molecule, while other polyphenols surely intervene in A. citrodora anti-AFB1 activity.