Role of Acidithiobacillus ferrooxidans culture in bioacidification and dewaterability of anaerobically digested sewage sludge (ADS) was investigated. A. ferrooxidans culture grown in 9K medium along with Fe(2+) produced iron flocculant containing, secondary iron minerals and biopolymeric substances as confirmed by FT-IR, XRD, and SEM-EDX. Bioacidification of ADS was performed using 10% (v/v) A. ferrooxidans culture, isolated cells and cell-free culture filtrate; and dewaterability was assessed using the capillary suction time (CST) and specific resistance to filtration (SRF). Isolated bacterial cells significantly (P<0.05) reduced the sludge dewaterability when supplemented with Fe(2+) while the whole culture and cell-free filtrate rapidly acidified the sludge without Fe(2+) and showed significant reduction of CST (71.3-73.5%) and SRF (84-88%). Results clearly indicated that the culture and filtrate of the A. ferrooxidans facilitated rapid sludge dewaterability while the cells supplemented with Fe(2+) also enhanced dewaterability but required 2-4days.