PMMA is the most commonly used denture base material, however some concerns regarding its biocompatibility have been heavily studied over the years. ACETAL and PEEK are more recently introduced denture base material alternatives which are believed to be more biocompatible than PMMA. Aim of the study: to evaluate and compare the short-term effects of PMMA, ACETAL and PEEK on human fibroblast cell line regarding cytotoxicity via direct contact. The assessment of cell cycle progression, cell death and ROS release were used as indicators. Materials and methods: WI-38 cell line was divided equally into four groups. Group I was the control group, for groups II, III and IV the WI-38 cells were cultured with PMMA, ACETAL and PEEK, respectively. The groups were all assessed by flowcytometry for cell cycle and cell death evaluation and ELISA for determining ROS after 4 days of culturing. Results: Pairwise analysis Bonferroni Method showed that highest mean values of total cell death, ROS levels and cell cycle arrest in the G2/M phase of the cell cycle was noted in group II (PMMA/WI38). Pearson`s correlation test (r test) showed that ROS levels correlated positively with total cell death and G2/M arrest. On the other hand, it correlated negatively with all the other parameters (G0/G1 and S). Conclusion: From the results of this study, we conclude that PEEK showed significantly superior biocompatibility compared to ACETAL and PMMA, from enough to justify its use as a denture base material in spite of its high price.