Abstract: The aim of this research was to develop a chalcone-based endodontic irrigant for cleaning and disinfecting the root canal. Minimal inhibitory concentration (MIC) experiments in C. albicans and E. faecalis strains with different aminochalcones (AM) were carried out, and the compound that presented the best activity against both pathogens was chosen. The formulation of an endodontic irrigant was elaborated, tested in mono and dual specie biofilms. Disks were sterilized and then incubated with E. faecalis, C. albicans and E. faecalis and C. albicans mixed for 72 h for biofilm maturation. After contamination, samples were divided in 4 experimental groups and 2 positive control group as follows: Group1: Irrigant; Group2: Irrigant + AM-38; Group3: Chlorhexidine 2% (positive control) and, Group 4: 1.0% sodium hypochlorite (positive control). The samples were analyzed by CFU/ml count. The sample was taken to sonicador to remove the cells and then plated. The toxicity was determined in vitro with human gingival fibroblast cells (HGF) and in vivo using the Galleria mellonella model. Formulation showed antimicrobial activity, with MIC on C. albicans 15.6 and E. faecalis 7.8 µg/ml. Treatment with formulation in concentration 156 µg/ml significantly reduced mono or dual species biofilm formation and viability (p < 0.05). The results were significant against C. albicans and E. faecalis and did not show toxicity in cells and G. mellonella. In general, the formulation showed effective antibiofilm activity, significantly reducing microorganisms, opening paths in search of new endodontic irrigants Ver menos