Research on biofilms is predominantly made in in vitro contexts. However, in vivo observation of biofilms in human chronic infections shows distinct differences compared to in vitro biofilm growth. This could imply the use of an inadequate mental model both in research and healthcare practices. Drawing on knowledge from the cognitive sciences, we hypothesise that the predominance of in vitro research on biofilms is skewed towards a mental model promoting wrong inferences for researchers and healthcare professionals (HCPs) in the in vivo context. To explore the prevalence of such a mental model, we carried out a qualitative image analysis in which biofilm illustrations from a Google image search were coded for typical in vitro or in vivo characteristics. Further, to investigate potential misinformed and unhelpful clinical interventions related to biofilms, we conducted a quantitative questionnaire among HCPs. The questions were designed to test whether knowledge about in vitro biofilms was used in an in vivo context. This questionnaire was analysed through a chi-squared test. Most biofilm illustrations were consistent with the in vitro model. A statistical analysis of survey responses revealed that HCPs have adequate knowledge about biofilm but often respond incorrectly when asked to apply their knowledge to in vivo contexts. The outcome of this research points to a prevalent and consolidated mental model derived from in vitro observations. This model has likely been made dominant by HCPs’ frequent exposure to visual depictions in articles and presentations. The prevalence of the in vitro model sets up the possibility of erroneous claims when the in vitro model is inadequately applied to in vivo contexts. This has potential implications for HCPs working in fields involving biofilm, such as wound care treatment.