Ionomeric materials release different proportions of fluoride and aluminum. Their simultaneous effect on the acidogenicity and composition of S. mutans biofilm is unknown. Six cylindrical specimens of each material (Ketac-fil, Vitremer, Fuji-Ortho LC, F-2000, and Z-100) were incubated with S. mutans GS-5 in culture media containing 5% sucrose (w/v). The media were changed daily for seven days, during which the pH and concentrations of fluoride and aluminum were determined. Furthermore, the concentrations of these ions and insoluble polysaccharide were determined in the biofilm formed at the end of the experimental period. The results showed that all the materials tested released fluoride. However, Vitremer released the highest amount of aluminum and was the most effective in reducing the acidogenicity of S. mutans biofilms. It also significantly affected both biofilm formation and composition. Thus, this study suggests that aluminum released by ionomeric materials may enhance the biological effects of fluoride.