Objectives: Various approaches are available for pit and fissure sealing, including: the use of sealants, with or without mechanical preparation; the use of etching, with or without bonding; and the use of lasers as an alternative to mechanical preparation. The objective of this study is to evaluate pit and fissure sealing by comparing the retention and microleakage of sealants, between mechanical and Er:Yag laser enamel preparation. Methods: Sixty extracted sound third molars are classified into six groups: A, bur mechanical preparation and sealant application; B, bur mechanical preparation, etching and sealant; C, bur mechanical preparation, etching, bonding and sealant; D, laser mechanical preparation and sealant; E, laser mechanical preparation, etching and sealant application; F, laser mechanical preparation, etching, bonding, and sealant. Statistical analysis methods include Fisher’s exact test, a general linear model for one-way analysis of variance (ANOVA) of multiple comparisons, and Bonferroni multiple comparison tests. Results: All the groups showed dye microleakage beneath the sealants. Less microleakage was observed for those that used bur rather than laser, 41 versus 44 specimens, respectively. The number of specimens without microleakage decreased as follows: group E (24), group A (18), groups B and F (17), group C (14), and group D (5). Retention was 100% in all groups except group D. Conclusion: Mechanical preparation increases retention of sealants, especially when etching material is used; additionally, bonding can help the retention. The best technique is mechanical preparation via laser and subsequent use of etching, without bonding prior to application of the dental sealant.