Antibacterial modification of medical materials has already been developed as a potentially effective method for preventing device-associated infections. However, the thin layer generated, often less than 1 µm, cannot ensure durability for metal devices in constant use. A novel stainless steel surface with both a quick bacterial killing rate and durability has been developed by synthesizing Cu and a supersaturated phase (S-phase) using a new active screen plasma alloying technology. This paper investigated the microstructure of a multilayer (using EDS/WDS, SEM, TEM and XRD) and the viability of bacteria attached to biofunctional surfaces (using the spread plate method). The experimental results demonstrate that the plasma alloyed multilayered surface case consists of three sublayers: a nano-crystalline (Fe, Cr, Ni)3N deposition layer (∼200 nm), a unique Cu-containing face-centred cubic (f.c.c.) γ'-M4N (M=Fe, Cr, Ni, Cu) layer and a Cu/N S-phase layer. The thicknesses of the total treated case and the Cu-containing layers are 15 and 8 µm, respectively. Copper exists as substitutional atoms in the γ'-M4N (with a constant concentration of about 5 at%) and in the S-phase lattice (reduces from 5 to 0 at%). The crystal constant of the Cu/N S-phase layer ranged from 0.386 to 0.375 nm, which is expanded by γ from 4.4% to 7.5%. An effective reduction of 99% of Escherichia coli (E. coli) within 3 h was achieved by contact with the homogeneous Cu alloyed surface. No viable E. coli was found after 6 h (100% killed).