The present research study assessed the cell viability and cytotoxic effect of mineral tri-oxide aggregate (MTA), Tetric N-Bond Universal bonding agent, Theracal PT (pulpotomy treatment), and platelet-rich fibrin (PRF) as pulp capping agents on human dental pulp stem cells (hDPSCs). The cells were isolated from the pulp tissue of an extracted healthy permanent third molar. After four passages in Dulbecco’s Modified Eagle’s Medium, the primary cells were employed for the investigation. The test materials and untreated cells (negative control) were subjected to an Methylthiazol-diphenyl-tetrazolium (MTT) cytotoxicity assay and assessed at 24-, 48-, and 72-h intervals. The Wilcoxon matched-paired t-test and Kruskal–Wallis analysis of variance (ANOVA) test were applied (p < 0.05). PRF imparted the highest cell viability at 48 h (p < 0.001), followed by MTA, Theracal PT, and Tetric N-Bond. Similarly, PRF had the highest potential to enhance cell proliferation and differentiation (p < 0.001), followed by Theracal PT, MTA, and the bonding agent at the end of 24 h and 72 h, respectively. Finally, PRF sustained the viability of human primary dental pulp stem cells more effectively than Theracal PT and MTA; however, the application of a Tetric N-Bond as a pulp capping agent was ineffective.