<p>The aim of this in vitro study was to evaluate the antibacterial activity and the level of cytotoxicity of tricalcium silicate-based cement (TCS) when combined with various antibacterial agents and concentrations. Agar diffusion and cell culture assays were used to assess the antibacterial and cytotoxic effects, respectively. TCS, serving as the control group, was combined with five different antibacterial agents: benzalkonium chloride (BAC), cetrimide (CTR), titanium oxide (TiO₂), zinc oxide (ZnO), and stannous fluoride (SnF₂), each at five different concentrations (0.5%, 1%, 2%, 4%, and 7%). Antibacterial activity was tested against five bacterial species: <i>Streptococcus mutans</i>, <i>Lactobacillus acidophilus</i>, <i>Lactobacillus casei</i>, <i>Actinomyces odontolyticus</i>, and <i>Escherichia coli</i> using the agar diffusion test. The cytotoxicity of BAC and CTR was evaluated on fibroblasts and human dental pulp stem cells (hDPSCs) using MTT analysis and live/dead staining. The one-way Analysis of Variance (ANOVA) and Kruskal-Wallis test were used for statistical analysis at a significance level of (<i>p</i> &lt; 0.05). The antibacterial activity was significantly influenced by both the type of antibacterial agent and its concentration. Compared to the control group, BAC demonstrated the highest antibacterial activity against all bacterial species, producing inhibition zones up to 30&#xa0;mm, whereas the control group exhibited zones of approximately 10&#xa0;mm. CTR exhibited significantly higher antibacterial activity - up to 20&#xa0;mm - against <i>L. acidophilus</i>, <i>L. casei</i>, and <i>A. odontolyticus</i> at concentrations ≥ 2%. TiO₂, ZnO, and SnF₂ displayed significantly higher antibacterial activity against <i>S. mutans</i> and <i>A. odontolyticus</i>, also only at concentrations ≥ 2%. <i>S. mutans</i> was the least responsive to antibacterial agents, while <i>L. acidophilus</i> and <i>L. casei</i> were predominantly affected by BAC and CTR, and to a lesser extent by the other agents. BAC exhibited no cytotoxicity up to 1%, slight cytotoxicity at 2% and 4%, and moderate cytotoxicity at 7% against fibroblasts. In contrast, it induced slight cytotoxicity at all tested concentrations against hDPSCs. CTR showed no cytotoxicity at all concentrations against fibroblasts compared to the control group, but exhibited slight cytotoxicity up to 1%, and moderate cytotoxicity at 2% and higher concentrations against hDPSCs. Notably, hDPSCs were more susceptible to the cytotoxic effects of antibacterial agents than fibroblasts. Compared to the control group, both organic antibacterial agents (BAC and CTR) demonstrated superior antibacterial activity, whereas the inorganic agents showed significant antibacterial properties only at high concentrations. BAC outperformed CTR in antibacterial activity but was associated with higher cytotoxicity at elevated concentrations. An optimal balance between antibacterial efficacy and biocompatibility was achieved with TCS combined with 1% BAC, demonstrating strong antibacterial activity (up to 30&#xa0;mm inhibition zones), 100% viability in fibroblasts, and over 60% viability in hDPSCs.</p>