Objectives: Vertical root fractures (VRF) can be a major challenge in root canal treatment due to bacterial biofilms' colonization of the fractures. The aim of this study was to evaluate the histological bacterial penetration and proliferation after sealing the simulated VRF with different intracanal sealing materials using confocal laser scanning microscopy (CLSM). Materials and methods: Sixty extracted human single-rooted premolars were used, and VRFs were created after root canal preparation. The canals were filled with either mineral trioxide aggregate (MTA), gutta-percha (GP) with AH plus sealer, or GP with bioceramic (BC) sealer. Enterococcus faecalis bacterial infection was induced, and bacterial colonization was assessed in the dentinal tubules and simulated VRF. Results: In the MTA group, there were significantly more dead bacteria in the dentinal tubules than live bacteria. The AH group had significantly more live bacteria than dead bacteria, and there were significantly more bacteria in the AH group compared to the BC and MTA groups. The study also revealed that bacteria can colonize the simulated VRF, from the part of the fracture to the outer surface. Clinical relevance: Understanding the pathological process following VRF and evaluating the ability of the VRF filling to prevent bacterial migration through the filled root end is crucial. Furthermore, quantifying the exact routes of microbial colonization, at the filling-dentine interfaces, and into the dentinal tubules, can provide valuable information to clinicians for selecting the appropriate root canal filling material to minimize bacterial colonization and improve treatment outcomes.