Polydimethyldiallylammonium chloride (PDDA) is an excellent flocculant for wastewater purification and sludge dewatering, but whether it poses a threat to hydrogen production from waste activated sludge is not known. In this study, the effect and underlying mechanism of PDDA on the dark fermentation of sludge was investigated. The results showed that PDDA reduced cumulative hydrogen production from 3.8±0.1 to 2.4±0.1 mL/g volatile suspended solids at 40 g/kg total suspended solids. PDDA impeded the dark fermentation process by inhibiting the activity of key enzymes, presenting a stronger inhibitory effect on the hydrogen production process than the hydrogen consumption process. Additionally, PDDA inhibited Firmicutes by enriching other microorganisms, thereby impeding hydrogen production via the acetate pathway. This study deepens the understanding of the potential effects of PDDA on sludge treatment and provides a theoretical basis for alleviating the negative effects of quaternary ammonium-based cationic flocculants.