Four polymer and ceramic computer-aided design/computer-aided manufacturing (CAD/CAM) materials from different manufacturers (VITA CAD-Temp (polymethyl methacrylate, PMMA), Celtra Duo (zirconia-reinforced lithium silicate ceramic, ZLS), IPS e.max CAD (lithium disilicate (LS2)), and VITA YZ (yttrium-tetragonal zirconia polycrystal, Y-TZP)) were tested to evaluate the cytotoxic effects and collagen type I secretions on human gingival fibroblasts (HGFs). A total of 160 disc-shaped samples (Ø: 10 ± 2 mm; h: 2 mm) were milled from commercial blanks and blocks. Direct-contact cytotoxicity assays were evaluated at 24, 48, and 72 h, and collagen type I (COL1) secretions were analysed by cell-based ELISA at 24 and 72 h. Both experiments revealed statistically significant differences (p < 0.05). At 24 and 48 h of contact, cytotoxic potential was observed for all materials. Later, at 72 h, all groups reached biologically acceptable levels. LS2 showed the best results regarding cell viability and collagen secretion in all of the time evaluations, while Y-TZP and ZLS revealed intermediate results, and PMMA exhibited the lowest values in both experiments. At 72 h, all groups showed sharp decreases in COL1 secretion regarding the 24-h values. According to the results obtained and the limitations of the present in vitro study, it may be concluded that the ceramic materials revealed a better cell response than the polymers. Nevertheless, further studies are needed to consolidate these findings and thus extrapolate the results into clinical practice.