Autoinducer-2 (AI-2) is a cell-to-cell signaling molecule which is thought to be utilized for quorum sensing processes by a variety of prokaryotic species. This molecule is usually detected using a so-called autoinducer bioassay, which relies on the ability of a Vibrio harveyi reporter strain to produce light in response to AI-2. However, as previously reported, the presence of glucose in the sample can inhibit the bioluminescence of the reporter strain, a fact that is often ignored by investigators. Our data suggest that the presence of glucose in concentrations below that required for the inhibition of bioluminescence may lead to incorrect AI-2 readings and produce misleading (false-positive) results. Our findings also suggest that even if all the limitations of this bioassay are considered, the large standard deviation of the method allows only for a qualitative and not quantitative interpretation of the obtained results.