Bioinspired nanotopography has emerged as a promising approach to generate antimicrobial surfaces to combat implant-associated infection[1]. Although tremendous efforts have been made to develop bactericidal 1D (e.g., nanopillar) structures, the bactericidal capacity of 2D (e.g., nanoflake) structures remains uncertain[2]. Here, we utilized hydrothermal synthesis to generate 2D nanoflake surfaces on pure titanium substrates and investigated the physiological effects of nanoflakes on bacteria. Our results showed that nanoflakes impaired the attachment of Gram-negative bacteria such as Escherichia coli and triggered the production of reactive oxygen species (ROS), leading to killing of the adherent bacteria. Consequently, the mean thickness of biofilm was significantly suppressed on nanostructured surfaces. E. coli surface appendage type-1 fimbriae was not implicated in the nanoflake-mediated modulation of bacterial attachment but did influence the bactericidal effects of nanoflakes. An E. coli ΔfimA mutant lacking type-1 fimbriae was more susceptible to the bactericidal effects of nanoflakes compared to the parent strain, while fimA+ mutants showed higher bacterial viability but not fully restored to parent levels. These results suggest that bacteria-nanoflake interactions mediated by type-1 fimbriae modulate the efficacy of the antimicrobial properties of nanoflake surfaces against E. coli. This finding offers a better understating of bactericidal surfaces and can facilitate the design of antimicrobial surfaces in medical applications in the future.