Objectives: Pit and fissure sealants provide a protective barrier against microbial infiltration and plaque accumulation, effectively reducing the risk of occlusal caries. However, microleakage remains a significant challenge, influencing sealant efficacy. This study evaluates and compares the penetration depth and microleakage of hydrophilic sealants and flowable composites under in vitro conditions. Methods: Twenty extracted third molars with deep pits and fissures were randomly divided into two groups. Acid etching was performed using 37% orthophosphoric acid, followed by the application of either a hydrophilic sealant or a flowable composite. Samples were immersed in 1% Methylene blue dye for 24 hours to assess microleakage. After sectioning, penetration depth was measured using a stereo-microscope. Statistical analysis was conducted using SPSS software (Version 20.0, IBM Corp, Armonk, NY, USA) with the Mann-Whitney U test. Results: The hydrophilic sealant group exhibited significantly lower dye penetration and higher penetration depth percentages compared to the flowable composite group, indicating superior sealing properties and adaptability. Conclusion: Hydrophilic sealants demonstrated better performance in terms of reduced microleakage and enhanced penetration depth compared to flowable composites, making them a promising choice for fissure sealing, particularly in conditions with moisture challenges. Further studies are recommended to confirm these findings and assess long-term clinical performance..