Assessing the biocompatibility of endodontic root-end filling materials through cell line responses is both essential and of utmost importance. This study aimed to the cytotoxicity of the type of cell death through apoptosis and autophagy, and odontoblast cell-like differentiation effects of MTA, zinc oxide–eugenol, and two experimental Portland cements modified with bismuth (Portland Bi) and barium (Portland Ba) on primary cell cultures. Material and methods: The cells corresponded to human periodontal ligament and gingival fibroblasts (HPLF, HGF), human pulp cells (HPC), and human squamous carcinoma cells from three different patients (HSC-2, -3, -4). The cements were inoculcated in different concentrations for cytotoxicity evaluation, DNA fragmentation in electrophoresis, apoptosis caspase activation, and autophagy antigen reaction, odontoblast-like cells were differentiated and tested for mineral deposition. The data were subject to a non-parametric test. Results: All cements caused a dose-dependent reduction in cell viability. Contact with zinc oxide–eugenol induced neither DNA fragmentation nor apoptotic caspase-3 activation and autophagy inhibitors (3-methyladenine, bafilomycin). Portland Bi accelerated significantly (p < 0.05) the differentiation of odontoblast-like cells. Within the limitation of this study, it was concluded that Portland cement with bismuth exhibits cytocompatibility and promotes odontoblast-like cell differentiation. This research contributes valuable insights into biocompatibility, suggesting its potential use in endodontic repair and biomimetic remineralization.