Previous descriptions of quercetin, a widely studied flavonoid, as a frequently reported nonspecific screening hit due to aggregating behavior has raised questions about the reliability of in vitro bioactivity reports of phenolic compounds. Here a systematic study on 117 phenolic compounds is presented, concerning their aggregating tendency and the relevance of this phenomenon to obtaining false bioassay results. Fourteen compounds formed aggregates detectable by dynamic light scattering (DLS) when assayed at 10 µM in Tris-HCl pH 7.5. Flavonoids were more prone to aggregation than other phenolic compounds, and the aggregate formation was highly dependent on the vehicle, ionic strength and pH. The compounds were also assayed against three unrelated enzymes in the presence and absence of Triton X-100, and their bioactivity ratios were collected from PubChem database. By comparing these datasets, quercetin and rhamnetin were confirmed as promiscuous inhibitors. In general, flavonoids exhibited also higher bioactivity ratios in the PubChem database than coumarins or organic acids. To conclude, aggregate formation can be controlled with Triton X-100 and this phenomenon needs to be considered when bioassay data is interpreted, but our data indicates that it does not always lead to unspecific inhibition of biological targets.