Aim: Root canal irrigation is an essential step in the root canal treatment. The aim of this study was to compare the efficacy of three agitation methods [manual-dynamic irrigation gutta-percha (GP), EndoActivator, and passive ultrasonic irrigation (PUI)] for 2.5% NaOCl irrigation solution in debriding two organic films (collagen and hydrogel) from simulated root canal walls. Materials and methods: Twenty-four Endo-Vu blocks were used to create half root canals using Profile instruments. The working length was 0.5 mm short of the canal end. The canal was enlarged to 30/06. The canals were allocated into two experimental groups (n = 12): group I (collagen film models), group II (hydrogel layer models). Each group was subdivided into three equal subgroups (n = 4): manual-dynamic irrigation (GP), EndoActivator sonic irrigation, and PUI. Each half canal was then covered with a flat glass slide, before quantifying the collagen or hydrogel film by using the Image-Pro Plus 4.5 and ipWin4 software. The data were analyzed using the generalized linear mixed model. Results: The duration of irrigation had a significant influence on surface area coverage with the residual film (hydrogel, collagen). Exposure of the organic film to PUI irrigation was significantly associated with more film removal than GP irrigation (p = 0.0001). Exposure of the organic film to EndoActivator irrigation was significantly associated with more film removal than GP irrigation (p < 0.001). Conclusion: The protocol of EndoActivator agitation of the NaOCl irrigant is more effective in simulant biofilm removal than gutta-percha pumping but less effective than passive ultrasonic agitation. Clinical significance: The findings from this study make several contributions to enhance our understanding of the use of passive agitation of NaOCl, when compared to sonic and manual agitation as a final irrigation protocol, in the removal of the biofilm from the apical, middle, and coronal thirds of the root canal.