Metal surface characteristics play a significant role in interacting with their biological environment. Copper surfaces have been identified for their antimicrobial properties. Improvement of antibacterial and antiviral performances can be tailored by surface microstructure modification. Severe plastic deformation is an effective surface modification procedure to improve the mechanical performance of metal surfaces. This technique can be adapted to obtain surface grain refinement and induce surface roughness. In this work, cold spray shot peening is used to modify copper substrate surfaces and study the effects on their antibacterial properties. To modify the grain structure of copper, different shot-peening parameters were examined. The surface roughness and microstructure were investigated by employing optical and scanning electron microscopy. The bactericidal activity of copper substrates after shot peening treatment is discussed and a comparison between the bacterial load on treated (shot-peened surface and cold sprayed copper coating) and untreated surfaces (as-received) is provided. Testing of the surfaces after their exposure to the biological environment demonstrated improved microbial inactivation performances for surfaces that had undergone grain refinement without exceeding a certain roughness value.