Purpose: The aim of this systematic review and meta-analysis is to answer the focused question: Does the application of phosphoric acid, Er:YAG laser and air abrasion enamel conditioning methods previous to the oclusal sealant application in human permanent molars influence the microleakage? Study design: A literature research was carried out in the Pubmed Medline, Web of Science, Scopus and Cochrane databases using with the MeSH terms and keyword search strategy. A supplemental hand search of the references of retrieved articles was also performed. Inclusion criteria comprised ex vivo studies (extracted teeth) with permanent human teeth that used chemical (phosphoric acid) or mechanical (Er:YAG laser and air abrasion) conditioning methods previous the sealant application. The studies should evaluate microleakage as an outcome. Meta-analysis pooled plot were obtained comparing the microleakage after pre-treatment with phosphoric acid, Er:YAG and air abrasion enamel conditioning for sealant application using RevMan software. Results: The search resulted in 164 articles, 55 records were excluded because they were duplicated. The analysis of titles and abstracts resulted in the exclusion of 105 studies. Four studies were included in the systematic review and the meta-analysis. According to the risk of bias evaluation, the four studies were considered low risk of bias. The meta-analysis showed that phosphoric acid had lower microleakage than Er:YAG laser (p < 0.001) and air abrasion (p < 0.001), with heterogeinity of I2 = 0% and I2 = 71%, respectively. It was not found statistical difference when compared phosphoric acid and phosphoric acid combined with Er:YAG laser and air abrasion (p > 0.05). Conclusion: The evidence supports that the pretreatment with phosphoric acid leads lower microleakage in oclusal sealants than Er:YAG laser and air abrasion.