One of the factors affecting the success of endodontic treatment is to fill the root canal system hermetically. The aim of this in vitro study was to evaluate the effect of nonthermal plasma (NP) on dentinal tubule penetration of root canal sealers using confocal laser scanning microscopy. Forty mandibular premolar teeth were selected and the root canals were prepared with large-Waveone-Gold rotary-files. Specimens were divided into four experimental groups according to sealer and NP treatment (n = 10). G1: AH-Plus (AH) G2: nonthermal plasma application + AH-Plus(AH-P) G3: Endosequence-BC(BC) G4: nonthermal plasma application + Endosequence-BC(BC-P). Cold lateral-condensation technique was used for the obturation of root canals. The roots were sectioned horizontally and the sections were examined under confocal laser scanning microscopy. The maximum tubule penetration and percentage of penetration values were obtained from the microscopy images and were statistically analyzed with repeated measurements-ANOVA and the Tukey (HSD) test (p < 0.05). The percentages of dentinal tubule penetration of the groups were not statistically different. The maximum tubule penetration of the AH-P was statistically lower than that of the BC-P (p < 0.05). Plasma application had no affect on the percentage of dentinal tubule penetration. Under the conditions of this in vitro Endosequence-BC sealer showed higher maximum tubule penetration values than AH-Plus after NP treatment. Percentage of dentinal tubule penetration values of experimental groups was similar.