Per- and polyfluoroalkyl substances (PFASs) are a diverse class of aliphatic organofluorine-containing compounds that are widely used in a variety of commercial and industrial applications including as major constituents of fire-fighting foams, commercial products that require water- and grease-repellency, and industrial manufacturing. The broad utility of PFASs in commercial and industrial applications is linked to their unique chemical properties that provide thermal and chemical stability along with surfactant-like properties. These same properties result in PFASs being persistent in the environment leading to concerns about environmental and human health risks resulting from exposure to PFAS and the known potential of PFASs to bioaccumulate and exhibit toxic effects. Whereas perfluoroalkyl substances such as the perfluoroalkyl acids (PFAAs) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) have received significant attention from regulatory agencies, most PFASs in the environment are polyfluoroalkyl substances with less defined chemical structures. Previous studies demonstrate that many polyfluoroalkyl substances transform in the environment into terminal PFAAs. Therefore, polyfluoroalkyl substances are considered as so-called PFAA “precursors” and their transformations warrant investigation for the further development of environmental regulations and to guide PFAS remediation efforts. The overarching goal of this thesis is to elucidate the biotransformation rates, biotransformation products, and biotransformation pathways for a set of environmentally relevant PFAA precursors. To meet this goal, experiments were designed to explore the biotransformation of 15 diverse PFAA precursors in a self-consistent biotransformation test system seeded with wastewater microbial communities. Individual precursors were spiked into the biotransformation test system and samples were collected over 96 h and measured by means of high-resolution mass spectrometry (HRMS). The HRMS acquisitions were mined to quantify the disappearance of the parent compounds and estimate biotransformation rate constants, and to identify evidence of biotransformation product formation and propose structures of putative biotransformation products using an integrated suspect screening and nontarget analysis approach. The resulting dataset was carefully evaluated to propose comprehensive biotransformation pathways based on time-dependent abundance profiles of the putative biotransformation products. Several key results have emerged from this thesis. First, the average biotransformation rate constants estimated for the 15 parent compounds range over nearly four orders of magnitude, reflecting major differences in their biotransformation potential. Interestingly, the major differences in the biotransformation potential among the 15 selected parent compounds can be explained by systematic differences in their chemical structure. Second, structures of 144 putative biotransformation products are proposed for the 15 parent compounds in this thesis, 77 of which are unique chemical structures after accounting for replicate biotransformation products formed from two or more parent compounds. Based on a literature review performed at the time this thesis was written, 124 of these biotransformation products are identified here as products of one or more of the 15 selected precursors for the first time. Third, the proposed biotransformation pathways demonstrate that a few key biotransformations (e.g., monohydroxylations, dehydrogenations) are important steps in the biotransformation of many of the parent compounds to terminal PFAAs. Further, the data confirm that homologous parent compounds are biotransformed through homologous biotransformation pathways. Overall, the data in this thesis provide a comprehensive view on the aerobic biotransformation of a variety of PFAA precursors and the results can be used to support the development of biotransformation pathway prediction tools for PFAS.