Objective The aim of this study was to evaluate the effect of photodynamic therapy (PDT) and non-thermal plasma (NTP) on adhesion and sealer penetration in root canals.Material and Methods Sixty single-rooted premolars were used. The teeth were prepared using a crown-down technique. NaOCl and EDTA were used for irrigation and smear layer removal, respectively. The root canals were divided into three groups: control, PDT, and NTP. After treatments, the roots were filled using gutta-percha and either AH Plus (AHP) or MTA Fillapex (MTAF) sealers. Samples were sectioned at 4, 8, and 12 mm from the apex (1-mm slices)and analyzed by the push-out bond strength test (adhesion) and confocal laser scanning microscopy (sealer penetration). Data were statistically evaluated using Kruskal-Wallis, Dunn’s, and Spearman’s tests.Results Regarding AHP, bond strength was similar in the NTP group and in the control group, but significantly lower in the PDT group. As to MTAF, both therapies showed lower values than the control group. In the confocal analysis of AHP, maximum and mean penetration, and penetrated area were statistically higher in the control group than in the PDT and NTP groups. Penetrated perimeter was similar among groups. Regarding MTAF, all parameters yielded better results in the NTP than in the control group. The PDT and control groups showed similar results except for penetrated area.Conclusion PDT and plasma therapy affected the adhesion and sealer penetration of root canals filled with AH Plus and MTA Fillapex and there is no positive correlation between adhesion and sealer penetration.