Using direct current magnetron sputtering (DCMS), we generated flexible copper polyester surfaces (Cu-PES) and investigated their antimicrobial activity against a range of multidrug-resistant (MDR) pathogens including eight Gram-positive isolates (three methicillin-resistant Staphylococcus aureus [MRSA], four vancomycin-resistant enterococci, one methicillin-resistant Staphylococcus epidermidis) and four Gram-negative strains (one extendedspectrum β-lactamase-producing [ESBL] Escherichia coli, one ESBL Klebsiella pneumoniae, one imipenem-resistant Pseudomonas aeruginosa, and one ciprofloxacin-resistant Acinetobacter baumannii). Bactericidal activity (≥3 log10 CFU reduction of the starting inoculum) was reached within 15–30 min exposure to Cu-PES. Antimicrobial activity of Cu- PES persisted in the absence of oxygen and against both Gram-positive and Gram-negative bacteria containing elevated levels of catalases, indicating that reactive oxygen species (ROS) do not play a primary role in the killing process. The decrease in cell viability of MRSA ATCC 43300 and Enterococcus faecalis V583 correlated with the progressive loss of cytoplasmic membrane integrity both under aerobic and anaerobic conditions, suggesting that Cu-PES mediated killing is primarily induced by disruption of the cytoplasmic membrane function. Overall, we here present novel antimicrobial copper surfaces with improved stability and sustainability and provide further insights into their mechanism of killing.