In human risk assessment, bioavailability needs to be considered when relying on in vitro toxicity results. For single chemicals, this quantitative challenge is often handled through a bioavailability factor. For mixtures, however, things are more complicated. Thus, individual constituents may not only interact toxicodynamically and toxicokinetically, but the composition of constituents reaching the target site may also differ from what was present at the site of exposure due to the differences in their bioavailabilities. A recent study concluded on the in vivo potential of Australian tea-tree oil (TTO) to act as an endocrine disruptor based on an in vitro protocol measuring the growth of MCF-7 cells following chemical exposure to TTO. TTO is primarily used topically in humans, and is not a single chemical but is a mixture with some constituents penetrating the skin which others do not. The present study evaluated in an identical in vitro model to what extent TTO and its skin penetrating constituents affected the growth of MCF-7 cells. The estrogenic potency of TTO was confirmed, but none of the bioavailable TTO constituents demonstrated estrogenicity. The present study, therefore, cautions in vitro to in vivo extrapolations from the mixtures of constituents with potentially varying bioavailabilities.