The purpose of this study was to evaluate and compare the effect of erbium:yttrium-aluminum-garnet (Er:YAG) laser irradiation and conventional dental bur cavity preparation on in vitro microleakage of class V cavities restored with different adhesive restorative materials and two types of self-etching adhesives in primary teeth. Standard class V cavities were prepared on 80 extracted primary, and the teeth were randomly divided into eight subgroups prepared either by dental bur or Er:YAG laser irradiation and then restored with self-cured glass ionomer (GI), resin-modified glass ionomer (RMGI), resin composite and Clearfil SE Bond (two-step self-etching adhesive), and resin composite and Clearfil S3 Bond (one-step self-etching adhesive). Restorations were finished and stored in distilled water at 37 °C for 24 h and then subjected to thermocycling. All the teeth were sealed with nail varnish, placed in a silver nitrate solution, and then vertically cut in a buccolingually direction. Subsequently, the specimens were evaluated for gingival and occlusal microleakage using a stereomicroscope. Data were analyzed using Kruskal-Wallis test followed by Mann-Whitney test. Wilcoxon test was used for comparing occlusal microleakage with gingival microleakage at p < 0.05. A higher degree of occlusal and gingival microleakage values for the teeth restored with GI or RMGI was obtained by both preparation methods compared with that of resin composites and the two self-etching primers. Er:YAG laser irradiation resulted in a significantly higher degree of microleakage only at the gingival margins for teeth restored with GI or RMGI, or composite and Clearfil S3 Bond compared with the bur preparation. The Er:YAG laser-prepared teeth restored with composite and Clearfil SE Bond demonstrated a better marginal seal on occlusal and gingival margins compared with that of bur-prepared cavities. The degree of microleakage in class V cavities was affected by the type of adhesive restorative materials, type of self-etching adhesive, cavity margin location, and tooth preparation method either by Er:YAG laser or dental bur.