Biofilm contamination on an implanted medical device represents a particularity resilient reservoir of infection that inevitably leads to device failure. In this study, we demonstrate that an atmospheric pressure air plasma treatment can simultaneously eradicate biofilm contamination while beneficially functionalizing the underlying surface, creating long-lasting characteristics that inhibit microbial recolonization and promote fibroblast proliferation. By comparing two contrasting plasma treatments the interplay between plasma generated reactive species, biofilm contamination and the underlying surface was uncovered. The composition, wettability and topography of titanium surfaces were characterized using X-ray photoelectron spectroscopy, water contact angle measurements and atomic force microscopy. Exposure to plasma generated chemical species created nanoscale surface features and the introduction of oxygen and nitrogen containing functional groups, resulting in changes to the surface wettability. Using a polymicrobial biofilm model comprising of E. coli and S. epidermidis, it was shown that plasma can effectively eliminate biofilm contamination from the surface, while simultaneously functionalizing the surface to inhibit recolonization. To assess the biocompatibility of treated surfaces the adhesion and proliferation of murine fibroblasts was assessed using fluorescent microscopy, cell viability assays and flow cytometry. It was shown that plasma exposure led to surface characteristics that promote fibroblast adhesion and proliferation. Display omitted •Low temperature plasma simultaneously decontaminates and functionalizes titanium.•Plasma exposure creates nanoscale features and superhydrophilic characteristics.•Plasma exposure is highly effective in eliminating polymicrobial biofilms.•Plasma treated surface inhibits microbial recolonization and biofilm formation.•Biocompatibility improved through enhanced cell adhesion and proliferation.