Per- and polyfluoroalkyl substances (PFAS) are recalcitrant environmental contaminants whose biodegradation remains challenging due to the exceptional stability of carbon–fluorine bonds. Here, we employed Prospector® nanowell array technology to isolate synthetic bacterial communities from cotton detritusphere capable of degrading perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) as sole carbon sources. Screening of 96,000 nanowells across four inoculum-loading regimes identified high-density loading (20–33 genera per community) as the sole condition that yielded functionally stable consortia; low-, mid-, and overload-density regimes failed to support serial passaging functional stabilization. Eight communities (A–H) were characterized for PFAS degradation, fluoride liberation, and taxonomic composition via full-length 16S rRNA sequencing. Community H achieved 87.8 ± 5.6% PFOS removal within 10 days with near-stoichiometric fluoride release (2.93 μM; 97.7% theoretical maximum), while Community E achieved the highest PFOA removal (44.9 ± 21.8%). PFOS and PFOA degradation capacities were inversely correlated across communities, with Atlantibacter and Stenotrophomonas predicting PFOS performance (r = 0.70 and 0.50) and Enterobacter predicting PFOA performance (r = 0.80). Bioreactor-scale validation of Community H demonstrated 91.5 ± 1.4% PFOS removal under aerobic and 62.6 ± 4.7% under anaerobic conditions over 14 days. LC-MS/MS detected perfluorohexane sulfonate (PFHxS) as a transient intermediate, peaking at 147.7 ng mL⁻¹ (day 5, aerobic) before declining to <5 ng mL⁻¹ by day 13, confirming sequential C8 to C6 chain shortening. This work establishes nanowell array-guided community assembly as a reproducible strategy for developing PFAS bioremediation consortia and provides mechanistic evidence for growth-coupled defluorination without co-substrate amendment.