In this study, amorphous tantalum oxide coatings were deposited onto the Ti substrate with and without plasma electrolytic oxidation (PEO)-pretreatment by using a high-powered impulse magnetron sputtering system. The antibacterial tests were conducted using Syto9 fluorescent dyes to study the antibacterial performance of Gram-positive (Staphylococcus aureus) and Gram-negative (Actinobacillus actinomycetemcomitans) bacteria. In vitro studies, the cell cytotoxicity and cell viability were evaluated with soft-tissue derived human skin fibroblasts and hard-tissue derived human osteosarcoma cells. The results of the antibacterial experiments revealed that both the Ta2O5 and PEO-pretreated Ta2O5 samples had superior antibacterial properties compared to the uncoated Ti samples. Cell viability analyses showed that the Ta2O5 films with and without PEO pretreatment exhibited better cell growth compared to the Ti specimens in human skin fibroblasts and human osteosarcoma cells. The cell viability of PEO-pretreated Ta2O5 films even two-fold higher than the Ti specimens in human osteosarcoma cells. In addition, no cytotoxicity was detected in both the Ta2O5 and PEO-pretreated Ta2O5 films. It concluded that porous Ta2O5 film pretreated with PEO procedure possessed both great biocompatibility and antibacterial abilities. It might have highly potential to be a candidate for the surface materials of dental or orthopedic implants.