Per- and polyfluoroalkyl substances (PFASs) with varying chain lengths, headgroups, and alternative structures are widespread and persistent in soil, yet knowledge of their biological effects and toxic mechanisms on soil microorganisms is limited. This study identified the structure-dependent impacts of PFAS on microbial fatty acid (FA) profiles by integrated field-laboratory investigations. The field investigation uncovered distinct PFAS-FA interaction patterns, varying by PFAS fluorocarbon (FC) chain length and functional group, and FA hydrocarbon (HC) chain length and unsaturation degree. Specifically, long-chain perfluoroalkyl carboxylic acids (PFCAs, FC7-17) increased medium/long-chain FAs (HC ≤ 17) and monounsaturated FAs (MUFAs), while long-chain perfluoroalkyl sulfonic acids (PFSAs, FC6-8) enhanced longer-chain FAs (HC >
17) and MUFAs. Additionally, perfluoroether carboxylic acids (PFECAs) as well as short-chain PFCAs (FC <
7) and PFSAs (FC <
6), which commonly used as alternatives to long-chain PFCAs/PFSAs, were associated with polyunsaturated FAs (PUFAs). The laboratory investigation with