Aim: Little is known about the influence of isthmus morphology on the debridement efficacy of activated irrigation. The aim of this study was to investigate the influence of isthmus morphology on the debridement efficacy of laser-activated irrigation (LAI), EDDY and needle irrigation (NI), and to explain the methods of isthmus cleaning by LAI and EDDY. Methodology: Four root canal models (apical diameter: 0.30 mm, taper: 0.06, curvature: 23°, length: 20 mm) were produced by CAD-CAM with different isthmus morphologies: long-wide (4 mm; 0.4 mm), long-narrow (4 mm; 0.15 mm), short-wide (2 mm; 0.4 mm) and short-narrow (2 mm; 0.15 mm). The isthmuses were filled with a hydrogel containing dentine debris. The canals were filled with irrigant and models were assigned to the following irrigation protocols (n = 240): needle irrigation (NI) with a 30G needle, Eddy, and LAI (2940 nm Er:YAG-laser, 15 Hz, 40 mJ, SWEEPS, tip at the canal entrance). Standardized images of the isthmuses were taken before and after irrigation, and the amount of removed hydrogel was determined using image analysis software and compared across groups using Kruskal-Wallis test followed by Dunn's multiple comparison. Visualization of the isthmus during activation was achieved using a high-speed camera. The pattern and speed of the flow in the isthmus as well as transient and stable cavitation were analysed using imaging software. Results: Laser-activated irrigation, EDDY and NI removed more hydrogel in short-wide isthmuses than in narrow isthmuses (p < .001). LAI and EDDY removed more hydrogel than NI in every isthmus configuration (p < .001). EDDY showed eddies and stable cavitation, and LAI showed transient cavitation at each pulse, and pulsed horizontal flow with the highest particle speed in closed short isthmuses. Conclusions: Isthmus morphology influences debridement in all irrigation groups. Short-wide isthmuses were the easiest to clean while narrow isthmuses were the most challenging to clean. Width seems to be a more critical anatomical parameter than length. LAI and EDDY resulted in the greatest biofilm removal and performed better than NI. EDDY produced eddies and stable cavitation in the isthmus, and LAI showed transient cavitation and pulsed horizontal flow.