Objectives: This study evaluated the dentinal penetration depth of 2.5% sodium hypochlorite (NaOCl) in root canals with and without preparation and different irrigant activation protocols. Materials and Methods: Sixty-three bovine mandibular incisors were randomly allocated to 6 groups (n = 10): G1, preparation + conventional needle irrigation (CNI); G2, preparation + passive ultrasonic irrigation (PUI); G3, preparation + Odous Clean (OC); G4, no preparation + CNI; G5, no preparation + PUI; G6, no preparation + OC; and CG (negative control; n = 3). Samples were filled with crystal violet for 72 hours. Irrigant activation was performed. Samples were sectioned perpendicularly along the long axis, 3 mm and 7 mm from the apex. Images of the root thirds of each block were captured with a stereomicroscope and analyzed with an image analysis software. One-way analysis of variance, followed by the Tukey post hoc test, and the Student’s t-test were used for data analysis, with a significance level of 5%. Results: The NaOCl penetration depth was similar when preparation was performed, regardless of the method of irrigation activation (p > 0.05). In the groups without preparation, G6 showed greater NaOCl penetration depth (p < 0.05). The groups without preparation had a greater NaOCl penetration depth than those with preparation (p = 0.0019). Conclusions The NaOCl penetration depth was similar in groups with root canal preparation.Without root canal preparation, OC allowed deeper NaOCl penetration. The groups without preparation had greater NaOCl penetration than those undergoing root canal preparation.