Rheometry is an experimental technique widely used to determine the mechanical properties of biofilms. However, it characterizes the bulk mechanical behavior of the whole biofilm. The effects of biofilm mechanical heterogeneity on rheometry measurements are not known. We used laboratory experiments and computer modeling to explore the effects of biofilm mechanical heterogeneity on the results obtained by rheometry. A synthetic biofilm with layered mechanical properties was studied, and a viscoelastic biofilm theory was employed using the Kelvin–Voigt model. Agar gels with different concentrations were used to prepare the layered, heterogeneous biofilm, which was characterized for mechanical properties in shear mode with a rheometer. Both experiments and simulations indicated that the biofilm properties from rheometry were strongly biased by the weakest portion of the biofilm. The simulation results using linearly stratified mechanical properties from a previous study also showed that the weaker portions of the biofilm dominated the mechanical properties in creep tests. We note that the model can be used as a predictive tool to explore the mechanical behavior of complex biofilm structures beyond those accessible to experiments. Since most biofilms display some degree of mechanical heterogeneity, our results suggest caution should be used in the interpretation of rheometry data. It does not necessarily provide the “average” mechanical properties of the entire biofilm if the mechanical properties are stratified.