Patients with orthopaedic implants often experience implant-associated infections (IAI), one of the most prominent causes for implant failure. IAIs are caused by pathogens adhering to the implant surface. One method to prevent IAIs is through the use of surface topography modifications. Cellular behaviour has been shown to be influenced by substrate surface topographies. Ideally, a topography that can simultaneously kill bacteria and promote osseointegration is desirable. In previous research, proved-to-be-bactericidal nanopillars of ~180 nm in height and ~80 nm in diameter were fabricated at 170 nm interspace, resulting in a bactericidal efficiency of 36 ± 5% for Staphylococcus aureus (S. aureus). The current research aimed to enhance this bactericidal efficiency by producing nanopatterns with different interspace distances and introducing disorder, while keeping the same nanopillar dimensions. The patterns have been produced by using electron beam induced deposition (EBID) at 100, 170, 300 and 500 nm interspacing. A disordered version of the 170, 300 and 500 nm samples was included with 45, 110 and 210 nm disorder distance, respectively. In order to keep the nanopillar dimensions consistent, EBID parameters were systematically optimised. The nanopatterns were interfaced with S. aureus cells and the bactericidal efficiencies were determined by counting the number of healthy, deformed and dead bacterial cells. For the ordered 100, 170, 300 and 500 nm interspace nanopatterns, bactericidal efficiencies of 62.3 ± 23.1%, 45.0 ± 31.4%, 8.6 ± 4.2% and 3.7 ± 2.3% were found, respectively. For the disordered 170, 300 and 500 nm interspace nanopatterns, bactericidal efficiencies of 45.9 ± 29.2%, 14.7 ± 7.2% and 12.7 ± 9.8% were found, respectively. These results showed a significantly higher bactericidal effect on lower-interspace nanopatterns. Disordered nanopatterns showed only slightly higher bactericidal efficiencies when compared to the ordered nanopatterns. This data indicated that bactericidal efficiencies could be increased relative to the previous nanopattern design. The findings of this research aid in developing antibacterial nanopatterns that are capable of killing gram-positive bacteria and support the path to prevention of IAIs.