The antibacterial nature of copperCopper and copperCopper alloys is well established, but our understanding of the precise mechanisms of toxicity continues to evolve. Few studies have focused on how the interaction of proteins, bacteria, and buffer constituents at the copperCopper/buffer interface can influence the antibacterial response, particularly for short-duration contact killing. The Advanced Small Drop Assay (ASDA) was used to compare antibacterial testing with phosphate-buffered saline (PBS), commonly used as a diluent in antibacterial testing, versus chloride-free Butterfield’s phosphate bufferPhosphate Buffers (BPB). Bacteria were more rapidly killed in PBS than in BPB when exposed to the copperCopper surfaces. Although both buffers produced nanoflowers, these nanostructuresNanostructures are different when exposed to PBS or BPB and have only limited effects on measured antibacterial responses. Etched copperCopper surfaces increased the antibacterial effects in the PBS and BPB buffers. These results indicate the importance of the effects of buffer composition on short-duration antibacterial testing, the utility of the ASDA to increase the throughput of antibacterial testing, and provide clues that will help resolve the mechanism(s) of toxicity.