Recent progress in nano-/micro-fabrication techniques has paved the way for the emergence of synthetic bactericidal patterned surfaces that are capable of killing the bacteria via mechanical mechanisms. Different design parameters are known to affect the bactericidal activity of nanopatterns. Evaluating the effects of each parameter, isolated from the others, requires systematic studies. Here, we systematically assessed the effects of the interspacing and disordered arrangement of nanopillars on the bactericidal properties of nanopatterned surfaces. Electron beam induced deposition (EBID) was used to additively manufacture nanopatterns with precisely controlled dimensions (i.e., a height of 190 nm, a diameter of 80 nm, and interspaces of 100, 170, 300, and 500 nm) as well as disordered versions of them. The killing efficiency of the nanopatterns against Gram-positive Staphylococcus aureus bacteria increased by decreasing the interspace, achieving the highest efficiency of 62 ± 23% on the nanopatterns with 100 nm interspacing. By comparison, the disordered nanopatterns did not influence the killing efficiency significantly, as compared to their ordered correspondents. Direct penetration of nanopatterns into the bacterial cell wall was identified as the killing mechanism according to cross-sectional views, which is consistent with previous studies. The findings indicate that future studies aimed at optimizing the design of nanopatterns should focus on the interspacing as an important parameter affecting the bactericidal properties. In combination with controlled disorder, nanopatterns with contrary effects on bacterial and mammalian cells may be developed