Substrate grain structure and topography play major roles in mediating cell and bacteria activities. Understanding these cell-substrate interactions is critical to improve regenerative orthopedic biomaterials as the population of people with damaged and degrading bone continues to grow. It is also becoming overtly evident that biomaterials should exhibit antibacterial activity to resist infection without using antibiotics, to which bacteria are becoming increasingly resistant. In this work, cell- and bacteria-substrate interactions were investigated on two common (but very different) orthopedic biomaterials with the objective of finding common parameters that may improve the performance of all biomaterials. First, a newly-developed severe shot peening (SSP) treatment was performed on 316L stainless steel, inducing increased nanoscale surface roughness and a network of overlapping slip bands (contributing to surface work hardening and substantial nanoscale grain refinement). Separation of the effects of nanoscale surface roughness and grain size was achieved by performing a secondary grinding/polishing step to remove differences in roughness between sample groups. Experiments with cells and bacteria revealed that the expression of vinculin focal adhesion contacts from osteoblasts was inversely related to grain size, while the adhesion of gram-positive bacteria (S. aureus and S. epidermidis) was inversely related to nanoscale surface roughness. Separately, magnesium oxide nanoparticles (MgONPs) were integrated into poly-L-lactic acid (PLLA) sheets, both alone and in combination with hydroxyapatite (HA) NPs, resulting in PLLA nanocomposites with significantly improved mechanical properties for bone applications. While the adhesion and proliferation of osteoblasts increased considerably on substrates containing MgONPs, the well-known bactericidal activity of MgONPs was not achieved, owing to poor NP exposure on the polymer surface. Therefore, an electrophoretic deposition (EPD) procedure was developed to coat a thin layer of MgONPs onto the PLLA. The colonization of both gram-positive (S. aureus and S. epidermidis) and gram-negative (P. aeruginosa) bacteria significantly decreased as the applied EPD voltage increased. The proliferation of osteoblasts increased as the induced surface energy increased. Comparing these two substrates provides insights into the complex interactions governing the performance of orthopedic implants. Importantly, it was found that increased surface energy (obtained here by mechanical (e.g., SSP) or chemical methods (e.g., adding MgONPs)) increased the expression of adhesion-mediating proteins and improved cellular adhesion/proliferation necessary for improving orthopedic applications. Moreover, both approaches highlight the importance of creating nanoscale surface features towards decreasing bacteria functions.