There is a need for reliable models to predict the food web bioaccumulation and assess ecological and human health risks of per- and polyfluoroalkyl substances (PFAS). This present study presents (i) the development of novel mechanistic aquatic and terrestrial food web bioaccumulation models for PFAS and (ii) an evaluation of model performance using available laboratory and field data. Model predictions of laboratory-measured bioconcentration factors and field-based bioaccumulation factors of PFAS in fish were in good agreement with observed data as measured by the mean model bias (MB), representing systematic over- or under-estimation and the standard deviation of the MB, representing general uncertainty. The models provide a mechanistic framework for evaluating the combined effect of simultaneously occurring uptake and elimination processes and indicate food web-specific magnification of PFAS, with the highest degree of biomagnification occurring in food webs composed of air-breathing wildlife. Albumin-water, structural protein–water, membrane-water distribution coefficients, and renal clearance rate are among the most important model parameters. With further development and testing, these models may be useful for future PFAS screening and risk assessment initiatives and advance bioaccumulation studies of PFAS by providing a mechanistic framework for PFAS bioaccumulation.