This study aimed to investigate the effects of different chelation solutions on the penetration of resin-based and bioceramic root canal sealers into dentinal tubules using a device that assembles conventional microplate detection with automated digital microscopy. Eighty-four single-rooted teeth were prepared with 30.07 nickel-titanium instruments and divided into four groups of 21 according to the final irrigation solutions used: saline (control), ethylenediaminetetraacetic acid (EDTA), maleic acid (MA), and etidronic acid (HEBP). The samples were obturated with AH Plus, MTA Fillapex, or EndoSequence BC Sealer using the single-cone technique. The roots were then sectioned horizontally at 2, 6, and 10 mm and examined using a Cytation 5 reader and Gen5 software. Data were compared using two-way analysis of variance followed by univariate analyses and Tukey's multiple comparison test (p < .05). The mean penetration depth in the apical thirds was higher in the MA group than in the other groups (p = .013). The mean depth in the middle and coronal thirds was almost equal in the EDTA and HEBP groups. The mean depth was significantly higher in the coronal thirds of the root canals than in any other section in all final irrigation solution groups (p < .001). EndoSequence BC Sealer displayed higher penetration depth than AH Plus and MTA Fillapex, although the differences between the examined sealers were not statistically significant. The chelation solutions increased root canal sealer penetration into the dentinal tubule.