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Background: The effectiveness of ready-to-use disinfectant wipes was previously assessed in standardized suspension tests, which were inadequate because they ignored that the wipes are rubbed against a surface. Thus, we assessed the effectiveness of commercially available disinfectant wipes impregnated with an alcoholic solution according to the 16615 standard, which includes a test with mechanical action. Methods: According to the EN 16615 standard, under clean conditions, four squares (5 cm × 5 cm), placed next to one another, were marked on a test surface. Enterococcus hirae, Pseudomonas aeruginosa, and Staphylococcus aureus were inoculated on the leftmost square, and a wipe impregnated with an alcoholic solution was placed to the left of that square. Then, the wipe was pressed with a 2.5 kg weight and moved to the right and back to the left. After contact times of 1, 5, 10, or 15 min, we measured the reduction in bacterial load. Results: Alcohol-based ready-to-use commercial wipes did not show sufficient bactericidal activity at the contact times of 1, 5, 10 and 15 min. Wipes containing propan-1-ol and a mixture of propan-1-ol and propan-2-ol were active against Pseudomonas aeruginosa at the contact times of 1 min and 15 min. None of the examined wipes were active against Enterococcus hirae or Staphylococcus aureus. Conclusion: Bactericidal parameters of ready-to-use disinfectant wipes should be determined in surface tests, in addition to suspension tests, because suspension tests do not simulate the conditions under which disinfectant wipes are used in practice.