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Background: Pit and fissure sealing is an evidence-based preventive measure against dental caries, especially in primary teeth which possess high caries risk, and to give the required benefits of the sealant, a good adaptation, and sealing ability should be present. Purpose: This study aimed to assess and compare the microleakage score of Ionoseal® pit and fissure sealant, when used either alone or with prior surface treatment of erbium-doped yttrium aluminum garnet (Er: YAG) laser, acid etching, or their combination, in primary teeth. Materials and Methods: Forty healthy human molar teeth were randomly chosen and allocated into four study groups according to the surface pretreatment: group I, no surface pretreatment; Group II, 2W Er: YAG laser etching, Group III, combined laser etching and acid etching group, and Group IV, 37% phosphoric acid etching. After surface pretreatment procedures were conducted, the teeth were sealed with Ionoseal®. Subsequent microleakage was assessed through dye penetration under a stereomicroscope. One sample from each group was randomly selected, and scanning electron microscopy (SEM) was carried out on the central slice of the three obtained slices of the samples. Results: Chi-square test revealed a high statistically significant difference between groups (P = 0.00). Similarly, all pairwise comparisons revealed a statistically significant difference. Group I showed the greatest mean of microleakage score (1.5), followed by Group IV (1.4), Group II (0.7), and Group III which showed the least microleakage score (0.6). These findings were supported by the SEM examination results. Conclusions: Using Ionoseal with prior surface treatment by combined 2 W Er: YAG laser etching and 37% phosphoric acid etching can result in the best sealing ability which would significantly enhance the long-term success of pit and fissure sealing in primary teeth. [ABSTRACT FROM AUTHOR]