<p>The high antibiotic levels in antibiotic fermentation residues (AFR) impede their disposal or resource recovery due to their recalcitrance and toxicity, limiting organic matter degradation and methane production efficiency. This study has investigated the effects of varying-valence iron nanoparticles (Fe NPs) on the anaerobic Co-digestion (Co-AD) process of AFR and waste activated sludge (WAS), focusing on methane production, resistance gene expression, virulence factors, and microbial community interactions. The incorporation of varying-valence Fe NPs enhanced methane yield, with Fe³⁺ NPs increasing it by approximately 1.46-fold, effectively improving the biodegradation of recalcitrant bacterial residue. Fe³⁺ NPs facilitated microbial Fe(III) reduction under anaerobic conditions, thereby supporting enhanced electron transfer, redox activity, and methanogenic metabolism in the Co-AD system as evidenced by a 40.2% increase in cytochrome C. Microbiological analysis revealed that Fe³⁺ NPs upregulate genes associated with methanogenesis (frh, pta, mtr, and fwd), improving electron transfer between <i>Paraclostridium</i> and <i>Methanobacterium.</i> Additionally, Fe NPs induced enrichment of resistance genes and virulence factors, indicating microbial stress from oxidative and membrane perturbations, whereas Fe³⁺ NPs showed markedly weaker effects, superior biocompatibility, and the highest methane production. Fe NPs also promoted microbial metabolic activity and biofilm formation, optimizing Co-AD efficiency. Importantly, Fe³⁺ NPs exhibited better biocompatibility, with lower lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activities. By overcoming the limitations of conventional antibiotic fermentation residue treatment, this study highlights the potential of Fe NPs, particularly Fe³⁺ NPs, in enhancing anaerobic organics biomethanation and provides new insights into energy-efficient recalcitrant and toxic organic waste treatment strategies.</p>