The aim of the present study was to evaluate the effects of different surface pre-treatment techniques on the microleakage of a resin-based fissure sealant material. Thirty-five molars were divided into the following seven groups based on the surface pre-treatment technique used: (a) Erbium: Yttrium Aluminium Garnet (Er:YAG) laser (7 W); (b) Er:YAG laser (7 W)+acid etching (with 37% phosphoric acid); (c) Er:YAG laser (5.5 W); (d) Er:YAG laser (5.5 W)+acid etching; (e) air abrasion+acid etching; (f) air abrasion; and (g) conventional acid etching. The sealant was placed according to the manufacturers' instructions and light-cured for 20 sec. The sealed teeth were thermocycled for 10,000 cycles (5°-55°C), then immersed in 5% methylene blue for 24 h and sectioned mesiodistally. Each section was analyzed and photographed using a light microscope. Microleakage was assessed quantitatively by the degree of dye penetration and also qualitatively, scored on a three-point rating scale. Data were analyzed by one-way analysis of variance (ANOVA) and the Kruskal-Wallis test. The level of significance was set at p < 0.05. Regardless of the fissure morphology and penetration depth, enamel surfaces conditioned with Er:YAG laser irradiation exhibited significantly higher microleakage than those conditioned with air abrasion followed by acid etching, and those conditioned with conventional acid etching (p < 0.05). Air abrasion followed by acid etching, as well as conventional acid etching, provided a sufficient seal, whereas laser irradiation alone or in combination with acid etching exhibited higher microleakage than did the other groups. Conventional acid etching remains the most effective and the simplest technique.