Materials used for orthopedic implants should not only have physical properties close to those of bones, durability and biocompatibility, but should also exhibit a sufficient degree of antibacterial functionality. Due to its excellent properties, titanium is still a widely used material for production of orthopedic implants, but the unmodified material exhibits poor antibacterial activity. In this work, the physicochemical characteristics, such as chemical composition, crystallinity, wettability, roughness, and release of Ti ions of the titanium surface modified with nanotubular layers were analyzed and its antibacterial activity against two biofilm-forming bacterial strains responsible for prosthetic joint infection (Staphylococcus aureus and Pseudomonas aeruginosa) was investigated. Electrochemical anodization (anodic oxidation) was used to prepare two types of nanotubular arrays with nanotubes differing in dimensions (with diameters of 73 and 118 nm and lengths of 572 and 343 nm, respectively). These two surface types showed similar chemistry, crystallinity, and surface energy. The surface with smaller nanotube diameter (TNT-73) but larger values of roughness parameters was more effective against S. aureus. For P. aeruginosa the sample with a larger nanotube diameter (TNT-118) had better antibacterial effect with proven cell lysis. Antibacterial properties of titanium nanotubular surfaces with potential in implantology, which in our previous work demonstrated a positive effect on the behavior of human gingival fibroblasts, were investigated in terms of surface parameters. The interplay between nanotube diameter and roughness appeared critical for the bacterial fate on nanotubular surfaces. The relationship of nanotube diameter, values of roughness parameters, and other surface properties to bacterial behavior is discussed in detail. The study is believed to shed more light on how nanotubular surface parameters and their interplay affect antibacterial activity.