Background and Aim: Sealing occlusal fissure of posterior teeth is proved to be an effective approach in prevention of fissure caries. The aim of this in vitro study was to assess the level of microleakage at the sealant/tooth interface of four differently prepared enamel surfaces using Er, Cr:YSGG laser energy with and without additional routine acid.Materials & Methods: A total of 60 sound extracted premolars were included in this in vitro investigation. Samples were randomly assigned to four groups of control (using acid etching technique), laser alone Er, Cr: YSGG Laser radiation with power of 2w, RR: 20 HZ, Pd: 7601ls, and a supplemental etching before lor after laser application in 3rd and 4th Groups, Respectively. Prepared fissures were then sealed using Clin-pro TM sealant (3M, USA). Cases were then thermo cycled for 500 cycles in 5oC and 55oC followed by immersion of Subjects to a 1% methylene blue dye for 48 hrs. This was to enable microleakage assessment. Collected data was then analyzed using Kruskal-Wallis test.Results: No significant differences were found between the classical preparation techniques (Acid etching) and other three experimental groups. The degree of dye Penetration was found to be at its lowest level in the laser-prepared group alone followed by acid etching alone. However further laser intervention of acid etched surfaces was proved beneficial.Conclusion: Additional use of acid etching preparation after Er, Cr: YSGG laser application was shown to be superior to the individual use of acid or laser for enamel preparation, as the result of this study indicated a lower microleakage level on samples treated this way.