Objective: The aim of this study was to research the antimicrobial activity of fluoride-releasing dental materials on strains of mutans streptococci using a new in vitro method. Method and materials: Using microplaques for the cell cultures, specimens were made with a conventional glass ionomer (Ketac-Cem), a photo-cured ionomer (Vitremer), and a compomer (Dyract). Three groups were made, each focusing on a distinct situation: In the first group, the antimicrobial activity was tested after curing; in the second, the materials were exhausted from their disposable fluoride for 14 days; and in the third group, fluoride was reincorporated into the specimens after a topical application of 2% sodium fluoride gel for 4 minutes. Results: The results demonstrated that the photo-cured ionomer had the best antimicrobial activity of all groups, followed by the conventional glass ionomer and compomer groups, respectively. All materials studied presented antimicrobial activity in all stages of the experiment, which was statistically proven using the Student's t test. Conclusions: The fluoride-releasing materials studied in the current report demonstrated antimicrobial activity on strains of mutans streptococci in every group studied. This new method demonstrated more accuracy and practicability than others commonly used.