Poor osseointegration and infection are among the major challenges of 316L stainless steel (SS) implants in orthopedic applications. Surface modifications to obtain a nanostructured topography seem to be a promising method to enhance cellular interactions of 316L SS implants. In this study, arrays of nanodimples (NDs) having controlled feature sizes between 25 and 250 nm were obtained on 316L SS surfaces by anodic oxidation (anodization). Results demonstrated that the fabrication of NDs increased the surface area and, at the same time, altered the surface chemistry of 316L SS to provide chromium oxide- and hydroxide-rich surface oxide layers. In vitro experiments showed that ND surfaces promoted up to a 68% higher osteoblast viability on the fifth day of culture. Immunofluorescence images confirmed a well-spread cytoskeleton organization on the ND surfaces. In addition, higher alkaline phosphate activity and calcium mineral synthesis were observed on the ND surfaces compared to non-anodized 316L SS. Furthermore, a 71% reduction in () and a 58% reduction in () colonies were observed on the ND surfaces having a 200 nm feature size compared to non-anodized surfaces at 24 h of culture. Cumulatively, the results showed that a ND surface topography fabricated on 316L SS via anodization upregulated the osteoblast viability and functions while preventing and biofilm synthesis.