Objectives Evidence about modifications of dental luting materials to minimize biological failure at the "marginal gap" between teeth and fixed prosthodontics is scarce. We compared a copper-modified (Co-ZOP) and a conventional zinc oxide phosphate cement (ZOP) in terms of antimicrobial and cytotoxic potentials in vitro and in vivo. Materials and methods Specimens of ZOP and Co-ZOP were characterized by the mean arithmetic roughness (Ra) and surface free energy (SFE). Powder components were examined using scanning electron microscopy (SEM). Energy-dispersive X-ray spectroscopy (EDX) showed elemental material compositions. In vitro microbial adhesion was shown using SEM, luminescence, and fluorescence assays. CCK-8 assays of mouse fibroblasts (L929) and human gingival fibroblasts (GF-1) were performed after 6, 24, and 48 h of specimen incubation. In vivo, ZOP and Co-ZOP specimens were applied intraorally for 12 h; biofilm accumulation was shown using SEM. Results Ra of ZOP and Co-ZOP showed no significant differences; SFE was significantly higher for Co-ZOP. EDX exhibited minor copper radiation for Co-ZOP, none for ZOP. In vitro fungal adhesion to Co-ZOP was significantly higher than to ZOP; in vitro streptococcal adhesion, cytotoxicity, and in vivo biofilm formation were not significantly different. Conclusions Co-ZOP showed low surface allocations of copper with no improved antimicrobial properties compared with conventional ZOP in vitro or in vivo.