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Metallic implants are susceptible to bacterial colonization even years after the implantation impairing the osseointegration process. The treatment of a colonized implant is highly demanding, and in most cases implant replacement is the only effective solution. To avoid the bacterial attachment and proliferation, bactericidal coatings are proposed as a long-term prevention tool. Those coatings must assure a bactericidal activity for a long period and cannot induce cytotoxic responses in eukaryotic cells. Among all the bactericidal agents, Zinc is one of the most investigated due to its broad bactericidal activity spectrum and its stimulatory effect on bone formation. The aim of this study is to obtain a titanium oxide coating containing Zinc and evaluate its bactericidal activity, cytotoxicity and ion release profile. The coating was obtained by Plasma Electrolytic Oxidation (PEO) on commercially pure titanium grade 4 at 350 V for 60 s. Samples were divided in two groups; the reference group was obtained in a base electrolyte containing calcium acetate and calcium glycerophosphate (called CaP group). The experimental group had Zinc acetate added as a Zinc source to the base electrolyte (called Zn-CaP group). The surface was characterized by Scanning Electron Microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS), while the ion dissolution was evaluated by Inductively Coupled Plasma - Atomic Emission Spectroscopy (ICP-AES). The bactericidal activity was determined against Staphylococcus aureus by fluorescence microscopy using a live/dead viability kit. The cytotoxicity against eukaryotic cells was evaluated using adipose derived stem cells (ADSC) using the lactate dehydrogenase (LDH) assay. Zinc, Calcium and Phosphorous were incorporated to the titanium oxide coating and no changes on the coating structure and morphology were observed by the addition of Zn to the electrolyte. ICP-AES results show the coatings released Ca, P and Z ions after 28 days of immersion in DI water. The ICP-AES profile suggests the ion release reach an equilibrium state after 7 days of immersion. The Zn-CaP coating presented bactericidal activity against S. aureus, showing a higher number of dead bacteria after 6 h of incubation and a lower number of living bacteria after 24 h compared to the CaP group. No cytotoxic effect was observed against ADSC by the presence of Zn on the coating, indicating the Zn-CaP coating has a potential to prevent bacterial colonization in metallic implants.