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Bacterial infection of implanted biomaterials is a serious problem that increases health care costs and negatively affects a considerable fraction of orthopedic procedures. In this field, magnesium oxide nanoparticles (MgO NPs) have emerged as a promising material to combat bacterial infection while maintaining or improving bone cell functions. Here, MgO NPs were electrophoretically deposited onto poly‐L‐lactic acid (PLLA) sheets to achieve a coating of highly exposed MgO NPs that directly influenced cell‐substrate interactions at short time scales. Samples were characterized for their surface chemistry, crystal structure, roughness, wettability, degradation characteristics, and their ability to support the growth of human fibroblasts and osteoblasts, as well as their resistance to colonization byStaphylococcus aureus,Staphylococcus epidermidis, andPseudomonas aeruginosa. In general, increasing the applied voltage during deposition increased the surface coverage of the coating and significantly decreased the colonization of all three bacterial strains (up to a 90% reduction). Furthermore,S. aureuscells that did attach onto substrates prepared at high voltages exhibited trademark signs of membrane damage and cell death. Importantly, MTS cell viability assays indicated that osteoblast adhesion increased with increasing deposition voltage, while fibroblast adhesion exhibited the opposite trend. Thus, although requiring more studies, this research provides the first evidence that MgO NP coatings prepared at relatively high voltages (120–150 V) may have the ability to resist bacterial colonization, promote bone cell attachment, and curb fibrous capsule formation. Therefore, it is recommended that this technology be further investigated and developed for numerous orthopedic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3136–3147, 2017.