Objectives:The aim of the present study was to compare the effect of enamel pre-treatment with: 37% phosphoric acid-etching or self-etching adhesive and Er:YAG Laser (2940nm) on shear bond strength, microleakage and penetration depth of a fluoride-releasing resin-based pit and fissure sealant applied to sound premolars.Methods: Seventy-two sound premolars were allocated randomly into 3 groups according to enamel pretreatment protocol. The experimental groups included: Group I: 37% phosphoric acidetching, Group II: self-etching adhesive, Group III: Er:YAG Laser treatment. Each group was divided into two subgroups (A: shear bond strength, B: microleakage and penetration depth). Shear bond strength test was performed using Universal testing machine. Microleakage and penetration depth were tested after thermocycling (500×, 5-55°C, dwell time: 30s) and dye penetration method. Samples were assessed under Stereo-microscope with a magnification of ×40. One-way ANOVA followed by Tukey's post hoc test was used to analyze data.Results: Shear bond strength and microleakage results showed that there was statistically significant difference among different groups while penetration depth results showed there was no statistically significant difference among all groups.Conclusions: Conventional acid etching was the best pretreatment method prior to resin-based fissure sealants in terms of bond strength and microleakage.