The aim of the present study was to study the effect of fluoride gel treatment on fluoride release and inhibition of acid production of Streptococcus mutans by different glass ionomer cements. Test slabs of four glass ionomer materials were fitted into the bottom of a test tube. A layer of S. mutans cells was centrifuged onto the test slabs, and the specimens were incubated for 4 h in 1.7% sucrose solution. Incubations were made using fresh, aged (29 d), aged and F-treated (1.25% F-gel), and aged, F-treated and aged samples (n = 15 per group). After each incubation, pH and F contents of the fluid phase were determined. The freshly mixed glass ionomer samples released large amounts of fluoride, and the pH fall in the fluid phase was significantly inhibited. For aged samples, the fluoride release decreased strongly and no inhibitory effect on acid production by S. mutans was seen. After application of fluoride gel, fluoride release and inhibitory effect were significantly higher than initially for all glass ionomer cements. In conclusion, all glass ionomer cements were able to take up fluoride and subsequently release it, which resulted in reestablishment of their antibacterial effect. The patterns of fluoride release and antibacterial action were virtually the same for conventional and resin-reinforced glass ionomer cements.