Purpose To compare the sealing ability and penetration of a bioactive material used as pit and fissure sealant to those of glass ionomer sealant. Methods This was an in vitro experimental study conducted on 20 permanent teeth. For Group I of ten teeth, ACTIVA BioACTIVE-Base/Liner was applied as a sealant on pits and fissures, and the remaining ten teeth of Group II were sealed using glass ionomer cement. After thermocycling, the apex of the teeth was sealed using composite resin and they were immersed in 1% methylene blue solution, buffered at pH 7 for 24 h. Longitudinal sections were obtained from each tooth for evaluating the sealing ability and penetration, using a binocular light microscope at 4 × magnification. The obtained data were subjected to analysis using the Chi-square test and independent t test. Results The comparison of the sealing ability and sealant penetration, between the two groups, showed statistically no significant difference (p = 0.104 and p = 1.0, respectively). Conclusion Bioactive material as a pit and fissure sealant, performed on par with glass ionomer sealant in terms of tested properties like sealing ability and penetration.