A chemical disinfectant against surface-associated bacteria typically uses carriers (e.g., glass disks) that are purposely contaminated with bacteria prior to disinfection. After disinfection, the bacteria are harvested by mechanically separating them from the carrier surface to form a suspension of cells in a dilution tube. Bacterial clumps in the tube are disaggregated using mechanical or chemical techniques, thereby creating a well-mixed suspension of single cells suitable for enumeration. Efficacy is quantified by comparing the viable cell count for a disinfected carrier to the viable cell count for sham-disinfected (control) carrier. A test is said to be biased (invalid) if the observed efficacy measure is systematically higher or lower than the true efficacy. It is shown here for the first time that the bias attributable to the harvesting and disaggregating steps of a disinfectant test can be measured. For some conventional biofilm harvesting and disaggregating techniques, laboratory checks showed either negligible bias or important bias, depending on the disinfectant. Quantitative bias checks on the harvesting and disaggregating steps are prudent for each combination of carrier material, microorganism, and disinfectant. The quantitative results should be augmented by microscopic examination of harvested disinfected and control carriers and of the disaggregated suspensions.