Polyfluoroalkyl phosphates (PAPs), a group of fluorotelomer alcohol (FTOH)-based surfactants commonly used in water- and grease-proof food contact paper, have been suggested as a direct source of human exposure to health-concerned perfluoroalkyl carboxylic acids (PFCAs). This study investigated factors affecting biotranformation of 6:2 polyfluoroalkyl phosphates (6:2 PAPs) by three known FTOH-degrading Pseudomonas strains (Pseudomonas butanovora, P. oleovorans, and P. fluorescens DSM 8341) under different co-substrate conditions and compared to that by activated sludge samples. The three pure strains transformed 6:2 PAPs into eight different per- and poly-fluoroalkyl carboxylic acids (PFCAs) and/or PFCA precursors. P. fluorescens DSM 8341 produced 5:2 sFTOH [CF3(CF2)4CH(OH)CH3] and P. oleovorans produced 5:2 ketone [CF3(CF2)4C(O)CH3] as the primary transformation product, respectively, with citrate having a minimal impact on the transformation. P. butanovora with lactate produced more diverse transformation products than those by any two strains. Activated sludge was more efficient at transforming 6:2 PAPs and produced more transformation products including PFHpA [CF3(CF2)5COOH] and PFPeA [CF3(CF2)3COOH], with 5:2 sFTOH as the most abundant product on day 30. The abundance of the alkane hydroxylase (alkB) gene related to alkane oxidation, the changes of total microbial population as well as their community structure in activated sludge during 6:2 PAPs biotransformation were also investigated.