The unpredictable biodegradation of fluorotelomer (FT)-based per- and polyfluoroalkyl substances (PFAS) causes complicated risk management of PFAS-impacted sites. Here, we have successfully used redundancy analysis to link FT-based precursor biodegradation to key microbes and genes of soil microbiomes shaped by different classes of carbon sources: alcohols (C2-C4), alkanes (C6 and C8), an aromatic compound (phenol), or a hydrocarbon surfactant (cocamidopropyl betaine [CPB]). All the enrichments defluorinated fluorotelomer alcohols (n:2 FtOH; n = 4, 6, 8) effectively and grew on 6:2 fluorotelomer sulfonate (6:2 FtS) as a sulfur source. The butanol-enriched culture showed the highest defluorination extent for FtOHs and 6:2 FtS due to the high microbial diversity and the abundance of desulfonating and defluorinating genes. The CPB-enriched culture accumulated more 5:3 fluorotelomer carboxylic acid, suggesting unique roles of Variovorax and Pseudomonas. Enhanced 6:2 FtOH defluorination was observed due to a synergism between two enrichments with different carbon source classes except for those with phenol- and CPB-enriched cultures. While the 6:2 fluorotelomer sulfonamidoalkyl betaine was not degraded, trace levels of 6:2 fluorotelomer sulfonamidoalkyl amines were detected. The identified species and genes involved in desulfonation, defluorination, and carbon source metabolism are promising biomarkers for assessing precursor degradation at the sites.