Aim: The study aimed to compare the sealing ability, flow characteristics, and dimensional stability of three different root canal sealers: AH Plus, MTA Fillapex, and Bio-C® Sealer. Materials and methods: In this study, 75 freshly extracted mandibular incisors with straight, single root canals were selected. To ensure consistency, the teeth were decoronated to a standardized length of 11.5 mm. The root canals were prepared to the working length using a size 60 K file, with continuous irrigation using a 2.5% sodium hypochlorite solution and normal saline. Following instrumentation, the smear layer was removed by treating the canals with a 17% ethylenediaminetetraacetic acid (EDTA) solution for one minute. The prepared specimens were then randomly divided into three groups of 15 teeth each, with obturation performed using one of three sealers: Bio-C® Sealer, AH Plus, or MTA Fillapex. All sealers were prepared according to the manufacturer's instructions. The collected data were analyzed using SPSS software. Results: The study evaluated the mean sealer penetration depth, flow characteristics, setting time, and solubility of three different endodontic sealers: Bio-C® Sealer, AH Plus, and MTA Fillapex. Among the tested materials, Bio-C® Sealer exhibited the lowest mean penetration depth (311.23 ± 49.45 μm), whereas AH Plus and MTA Fillapex showed significantly higher penetration depths of 483.22 ± 67.21 μm and 472.32 ± 43.22 μm, respectively. A statistically significant difference was observed (p < 0.005), indicating that Bio-C® Sealer had significantly lower penetration than the other sealers. In terms of flow characteristics, Bio-C® Sealer demonstrated a flow of 23.14 mm, a setting time of less than 201 minutes, and a solubility of 2.10%. AH Plus exhibited the highest flow among the sealers (30.11 ± 5.51 mm), a notably shorter setting time (7.02 ± 5.73 minutes), and a solubility of 1.8%. MTA Fillapex had a flow of 27.22 mm, a setting time of 111 ± 18.21 minutes, and the lowest solubility (1.11 ± 1.62%). Conclusion: Root canal sealers are essential in endodontic treatment, impacting both sealing ability and biocompatibility.