Extrusion-based printing enables simplified and economic manufacturing of surgical guides for oral implant placement. Therefore, the cytotoxicity of a biocopolyester (BE) and a polypropylene (PP), intended for the fused filament fabrication of surgical guides was evaluated. For comparison, a medically certified resin based on methacrylic esters (ME) was printed by stereolithography (n = 18 each group). Human gingival keratinocytes (HGK) were exposed to eluates of the tested materials and an impedance measurement and a tetrazolium assay (MTT) were performed. Modulations in gene expression were analyzed by quantitative PCR. One-way ANOVA with post-hoc Tukey tests were applied. None of the materials exceeded the threshold for cytotoxicity (< 70% viability in MTT) according to ISO 10993-5:2009. The impedance-based cell indices for PP and BE, reflecting cell proliferation, showed little deviations from the control, while ME caused a reduction of up to 45% after 72 h. PCR analysis after 72 h revealed only marginal modulations caused by BE while PP induced a down-regulation of genes encoding for inflammation and apoptosis (p < 0.05). In contrast, the 72 h ME eluate caused an up-regulation of these genes (p < 0.01). All evaluated materials can be considered biocompatible in vitro for short-term application. However, long-term contact to ME might induce (pro-)apoptotic/(pro-)inflammatory responses in HGK.