Background <p>Surface-mediated transmission of microorganisms may lead to severe healthcare or food industry related outbreaks, and antimicrobial surfaces are one of the means to stop or decrease the surface transfer of bacteria. Standardized test methods conducted under idealized conditions and traditionally used to assess the antimicrobial activity of such surfaces do not however accurately reflect the real-life conditions of surfaces used in dry environments and high-touch settings. Previously, we have designed the HydroTouch test, which uses a semi-dry microbial inoculum on a finger-mimetic hydrogel to enable reliable quantification of microbial transfer efficiency under real-use-like conditions on environmental surfaces. Here, the HydroTouch test was refined in terms of microbial inoculum density, post-transfer exposure time and environmental conditions during surface exposure to enable the determination of surface transfer and antimicrobial activity.</p> Results <p>By using metallic copper as a model antimicrobial material, we showed that exposure time after microbial transfer from the finger-mimetic hydrogel to the surface is critical: short contact periods result in insufficient antimicrobial activity, whereas prolonged exposure leads to increased surface kill over time due to desiccation, complicating the interpretation of the antimicrobial effect. Among four bacteria and yeast tested, only <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> showed sufficient surface transfer and viability on controls to enable their reliable use on the HydroTouch test. Organic soiling during surface exposure was shown to have notably enhanced surface transfer of bacteria but at the same time it also reduced the activity of copper surface. Interlaboratory comparison of the method carried out in four laboratories indicated 3–17% bacterial surface transfer in different labs, whereas the interlaboratory differences could be attributed to variability in relative air humidity during touch surface preparation and exposure. During 10&#xa0;min exposure a modest but significant reduction of 0.30–0.75 log₁₀ in viable cell count on copper was observed by the participating laboratories and also this parameter was clearly affected by humidity of the exposure environment.</p> Conclusions <p>The utility of the modified HydroTouch test for the determination of microbial surface transfer and antimicrobial activity across different laboratories was demonstated with organic soiling and air humidity being the decisive factors for its reproducibility.</p>