This in vitro study evaluated the sealing ability and microleakage of calcium silicate-based sealers compared to an epoxy resin-based sealer. One hundred twenty-five roots from anterior teeth were chemo-mechanically prepared and divided into four groups: AH Plus (AH), ProRoot MTA (PR), Medcem MTA (MC), and Total Fill BC Sealer/BC-coated gutta-percha (TF); n = 30. Confocal laser scanning microscopy was used to measure sealer penetration at three horizontal levels in 10 roots per group, while glucose leakage over 30 days was assessed in 20 roots. A lateral compaction technique was used for most groups, except for TF, which employed a single-cone method. Data were analyzed using Python with a Kruskal–Wallis test and Dunn’s post hoc test. TF showed significantly greater penetration in the coronal and middle sections, while PR had the least penetration in the apical section. PR exhibited the highest canal circumference penetration, especially compared to MC and TF. Glucose leakage increased over time in all groups, with TF showing the highest permeability after 30 days. Overall, calcium silicate-based sealers PR, MC, and TF performed similarly to the epoxy resin standard AH, with all groups exhibiting decreasing penetration from coronal to apical and increased leakage over time.