The organic toxicity of sludge in land applications is a critical issue; however, minimal attention has been given to the mechanism of toxicity formation during high-strength wastewater treatment. To investigate the relevant factors that contribute to sludge toxicity, synthetic wastewater with N,N-dimethylformamide (DMF) was treated in a sequential aerobic activated sludge reactor. The acute toxicity of sludge, which is characterised by the inhibition rate of luminous bacteria T3, is the focus of this study. Using an operational time of 28 days and a hydraulic retention time of 12h, the study demonstrated a positive relationship between the acute toxicity of sludge and the influent DMF concentration; the toxicity centralised in the intracellular and inner sections of extracellular polymeric substances (EPS) in sludge flocs. Due to increased concentrations of DMF, which ranged from 40 to 200mgL(-1), the sludge toxicity increased from 25 to 45%. The organic toxicity in sludge flocs was primarily contributed by the biodegradation of DMF rather than adsorption of DMF. Additional investigation revealed a significant correlation between the properties of the bacterial community and sludge toxicity.