Uptake of 19 per- and polyfluoroalkyl substances (PFAS), including C3–C14 perfluoroalkyl carboxylic acids (PFCAs), C4, C6, and C8 perfluoroalkyl sulfonates (PFSAs), and four emerging PFAS, was investigated in two mushroom species (Agaricus bisporus and Agaricus subrufescens) cultivated in a biogas digestate-based substrate. Accumulation of PFAS in mushrooms was low and strongly chain-length dependent. Among the different PFCAs, bioaccumulation factors (log BAFs) decreased from a maximum of −0.3 for perfluoropropanoic acid (PFPrA; C3) to a minimum of −3.1 for perfluoroheptanoate (PFHpA; C7), with only minor changes from PFHpA to perfluorotridecanoate (PFTriDA; C13). For PFSAs, log BAFs decreased from perfluorobutane sulfonate (PFBS; −2.2) to perfluorooctane sulfonate (PFOS; −3.1) while mushroom uptake was not observed for the alternatives 3H-perfluoro-3-[(3-methoxy-propoxy)propanoic acid] (ADONA) and two chlorinated polyfluoro ether sulfonates. To the best of our knowledge, this is the first investigation of the uptake of emerging and ultra-short chain PFAS in mushrooms, and generally the results indicate very low accumulation of PFAS.