Objectives To investigate the effectiveness of root canal irrigation with chitosan on the dislocation resistance of a root canal sealer (MTA Fillapex) in vitro, measured by the push-out bond strength test. Materials and methods Root canals of mandibular premolars (n = 57) were prepared using rotary files with 5.25% sodium hypochlorite as the irrigant during instrumentation. Following this, the specimens were randomly divided into three groups (n = 19) based on the final irrigant: group 1, 0.2% chitosan solution; group 2, 17% EDTA solution; group 3, saline. Three specimens from each group were analyzed using scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). The remaining specimens of each group were divided into two subgroups (n = 8) based on the method of agitation of the final irrigants (chitosan/EDTA/saline): subgroup A, sonic (Endoactivator, Dentsply Maillefer); subgroup B, no activation (control). After irrigation, all specimens obturated with a commercial mineral trioxide aggregate-resin hybrid sealer (MTA Fillapex, Angelus, Londrina, Brazil). Dislocation resistance was measured using the push-out bond strength test after 3 weeks. The data were analyzed using Kruskal-Wallis test (P = 0.05). Results Immaterial of the irrigant agitation, groups irrigated with chitosan showed significantly higher bond strength values than those irrigated with EDTA (P < 0.05). Groups irrigated with saline showed the least bond strength values (P < 0.05). When EDTA was used, sonic agitation significantly improved the bond strength of the sealer, compared to the control (P < 0.05). There was no significant difference between sonic agitation and the control when chitosan solution was used as the final irrigant (P > 0.05). The nitrogen/carbon ratio was significantly higher in chitosan groups compared to the control group (P < 0.05). Conclusion This study provides the first evidence that chitosan irrigation improves the dislocation resistance of MTA-resin hybrid root canal sealer, compared to EDTA and saline irrigation.