This study compared the sealing effectiveness of fissure sealants bonded to enamel with universal adhesive systems applied in self-etch (SE) or etch-and-rinse (ER) modes. Extracted human molars (n = 216), were randomly assigned into nine groups with respect to etching mode, adhesive system, and the sealant material used (Clinpro/3 M or Giomer-Based BeautiSealant/Shofu): (1) ER + Clinpro (Control); (2) SE/Single Bond Universal (SBU) + ClinPro; (3) ER + SBU + Clinpro; (4) SE/All Bond Universal (ABU) + Clinpro; (5) ER + ABU + Clinpro; (6) SE/Clearfil Universal Bond (CUB) + Clinpro; (7) ER + CUB + Clinpro; (8) SE + BeautiSealant Primer + BeautiSealant; (9) ER/BeautiSealant Primer + BeautiSealant. Following thermocycling (1000X) and storage in 0.5% basic fuchsin, microleakage was evaluated quantitatively on tooth sections using imageJ analysis toolkit, and the data were analyzed statistically. The ER Mode significantly reduced the microleakage of universal adhesives. The following statistical ranking was observed with regard to the sealing effectiveness: ER + SBU + Clinpro = ER + ABU + Clinpro = SE/SBU + Clivnpro = ER + CUB + Clinpro > SE/ABU + Clinpro = SE/CUB + Clinpro > ER + BeautiSealant Primer + BeautiSealant = ER + Clinpro (Control) = SE/BeautiSealant Primer + BeautiSealant. While the tested universal adhesives showed less microleakage on pre-etched enamel, the sealing effectiveness of SBU under SE mode was similar to all universal adhesives under ER mode. In both ER and SE modes, the sealing effectiveness of the giomer-based sealant was comparable to the control group.