The purpose of this study was to examine the microleakage of three different fissure sealants after they were aged by mechanical loading and thermocycling in vitro. To this end, a bonding agent (Prime & Bond NT) and three different fissure sealants (Clinpro, Helioseal F, Teethmate F1) were used, whereby microleakage was evaluated using a dye penetration method after mechanical loading and/or thermocycling. Sealant-treated teeth were allocated into four groups: mechanical loading (50,000 times), thermocycling (10,000 times), mechanical loading (50,000 times) + thermocycling (10,000 times), and one control group. For each fissure sealant, both experimental and control groups showed statistically significant differences in average microleakage score (p < 0.05). Further, for each fissure sealant, the highest average microleakage score was obtained in mechanical loading + thermocycling group. When comparison was done for each aging method, the average microleakage scores showed statistically significant differences among the three fissure sealants (p < 0.05). Based on the results of this study, it was also concluded that it is necessary to develop reliable in vitro test methods for dental materials.