Aim: The present in vitro study was taken to compare penetration depth of five different root canal sealers to radicular dentin by confocal laser scanning microscope. Materials and Methods: 100 extracted intact single- rooted human teeth were collected. Teeth were decoronated, shaping and cleaning was done with rotary file system till #25 number file with 0.06% taper. Half of the samples (n=50) were irrigated without ultrasonic agitation while other half (n=50) samples were irrigated with ultrasonically agitation. Teeth were obturated and divided into five groups on the basis of five different sealer used (Group A: Zinc oxide eugenol sealer (control group), Group B: Apexit Plus, Group C: AH Plus, Group D: MTA Fillapex, Group E: Bio-C sealer). Samples were sectioned horizontally at 2mm, 4mm and 6mm from the apical region and penetration depth was observed under confocal laser scanning microscope. Data obtained was statistically analysed using Independent t test and Tukey Test. Results: Among the groups, Bio- C sealer (Group E) revealed greatest penetration depth at 2mm and 4mm while zinc oxide eugenol (Group A) had the least penetration depth. Among the subgroups, samples irrigated with ultrasonic agitation showed rise in penetration depth of the sealers.