Spread of pathogenic microbes and antibiotic-resistant bacteria in health-care settings and public spaces is a serious public health challenge. Materials that prevent solid surface colonization or impede touch-transfer of viable microbes could provide means to decrease pathogen transfer from high-touch surfaces in critical applications. ZnO and Ag nanoparticles have shown great potential in antimicrobial applications. Less is known about nano-enabled surfaces. Here we demonstrate that surfaces coated with nano-ZnO or nano-ZnO/Ag composites are not cytotoxic to human keratinocytes and possess species-selective medium-dependent antibiofilm activity against Escherichia coli, Staphylococcus aureus and Candida albicans. Colonization of nano-ZnO and nano-ZnO/Ag surfaces by E. coli and S. aureus was decreased in static oligotrophic conditions (no planktonic growth). Moderate to no effect was observed for bacterial biofilms in growth medium (supporting exponential growth). Inversely, nano-ZnO surfaces enhanced biofilm formation by C. albicans in oligotrophic conditions. However, enhanced C. albicans biofilm formation on nano-ZnO surfaces was effectively counteracted by the addition of Ag. Possible selective enhancement of biofilm formation by the yeast C. albicans on Zn-enabled surfaces should be taken into account in antimicrobial surface development. Our results also indicated the importance of the use of application-appropriate test conditions and exposure medium in antimicrobial surface testing.