Aims: To analyse the biocidal efficacy of thermal sprayed copper surfaces. Methods and Results: Copper alloy sheet metals containing >60% copper have been shown to exhibit potent biocidal activity. Surface biocidal activity was assessed by epifluorescence microscopy. After 2-h exposure at 20°C in phosphate-buffered saline (PBS), contact killing of Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis by brass sheet metal and phosphor bronze was 3-4-times higher than that by stainless steel. SEM observations revealed that the surface membranes of both bacterial strains were slightly more irregular when exposed to brass sheet metal than stainless steel. However, when exposed to phosphor bronze coating, E. coli were 3-4 times larger with irregular membrane morphology. In addition, the majority of the cells were associated with spherical carbon-copper-phosphate crystalline nanostructures characteristic of nanoflowers. The membranes of many of the S. epidermidis exhibited blebbing, and a small subset was also associated with nanoflowers. Conclusions: Our data indicate that increasing the surface roughness of copper alloys had a pronounced impact on the membrane integrity of Grampositive and, to a lesser degree, Gram-negative bacteria. In the presence of PBS, carbon-copper-phosphate-containing nanoflowers were formed, likely nucleated by components derived from killed bacteria. The intimate association of the bacteria with the nanoflowers and phosphor bronze coating likely contributed to their nonreversible adhesion. Significance and Impact of the Study: Thermal spraying of copper alloys provides a strategy for the rapid coating of three-dimensional organic and inorganic surfaces with biocidal copper alloys. Our study demonstrates that the macroscale surface roughness generated by the thermal spray process enhances the biocidal activity of copper alloys compared with the nanoscale surface roughness of copper sheet metals. Moreover, the coating surface topography provides conditions for the rapid formation of organic copper phosphate nanocrystals/nanoflowers.