Sealer staining using rhodamine B dye to investigate the penetration depth of endodontic sealers was proven unsuitable for this purpose. This study aimed to investigate the sealer penetration depth into dentinal tubules by scanning electron microscopy (SEM). Root canals of 52 human upper central incisors were instrumented using the ProTaper Gold NiTi system (Dentsply Sirona, York, PA, USA) up to size F3. After irrigation with sodium hypochlorite and citric acid combined with ultrasonic activation, the root canals were either filled using the epoxy resin sealer AH Plus (Dentsply Sirona) or the calcium silicate-based sealer Total Fill BC Sealer HiFlow (TFHF, FKG Dentaire, La Chaux-de-Fonds, Switzerland) by warm vertical compaction. Root slices of 1 mm thickness were obtained at 2 to 3, 5 to 6 and 8 to 9 mm from the apex. The root slices were investigated for sealer penetration into the dentinal tubules using SEM according to four root quadrants (buccal, mesial, oral, distal). Statistical analysis was performed by the Kruskal-Wallis test (p = 0.05) as data were not normally distributed according to the Shapiro-Wilk test. AH Plus penetrated significantly deeper into the dentinal tubules compared to TFHF at each root level (p < 0.05). Dentinal sealer penetration was deeper in the bucco-oral direction compared to the mesio-distal direction. AH Plus penetrated deeper into dentinal tubules than TFHF. Warm vertical compaction exerting high pressure on the root canal filling material is not able to press sealers deep into dentinal tubules as penetration depth values did not exceed a mean of 110 µm in SEM.