Successful root canal treatment depends on adequate obturation with biocompatible and non-cytotoxic materials. This study evaluated the in vitro and in vivo biological characteristics of an experimental polydimethylsiloxane (PDMS)-based endodontic sealer and compared it with Silco^® and Sealapex^® cement. Human dermal fibroblasts (HDFa) were exposed to polydimethylsiloxane-based sealer eluates, Silco^® and Sealapex^®, at concentrations of 1:200, 1:100, 1:50, 1:1, and undiluted eluate (1×) for 24, 48, and 72 h, and they were subcutaneously implanted in Wistar rats for 15, 30, and 45 days. Cell viability exceeded 90% at 24–48 h and remained at 85% at the highest concentration after 72 h. Sealapex^® showed approximately 85% viability at 24 h, over 70% at 48 h, and remained below the cytotoxicity threshold at 72 h. Silco^® showed a marked reduction, with values approaching 50% at 24 h. At 48 and 73 h, Silco^® showed a significant reduction in cell viability. Histological analysis revealed only mild acute and chronic inflammation, with no statistically significant differences over time. These results indicate that the experimental sealant demonstrates favorable biological properties suitable for further clinical evaluation.