Bioleaching, the addition of bacteria to geological materials, has been applied to sludge to remove metals and improve upon sludge dewaterability. This paper investigates the effect of using different quantities of inoculum (bacteria) during bioleaching on sludge dewaterability. The analysis was based on bioleaching experiments conducted in a 20 L bio-reactor using different quantities of inoculum (20%, 10%, 5%, 2%, 0%). Changes in pH, oxidation reduction potential (ORP), capillary suction time (CST), specific resistance to filtration (SRF) and extracellular polymeric substances (EPS) were determined to gauge sludge dewatering. Results indicate that sludge dewaterability during the 2%, 10%, and 20% inoculum experiments declined through time. Decreased dewaterability is attributed to increases in the quantity of proteins and polysaccharides in slime EPS. Dewaterability improved during the 5% inoculum experiment, and reached a maximum when pH was 2.3. During this latter experiment, CST and SRF were reduced by 74% and 62%, respectively, in comparison to control conditions, while total EPS content decreased by 71%. The decrease in total EPS was primarily due to a decrease in proteins associated with tightly bound EPS (TB-EPS). Thus, changes in the amount of proteins in TB-EPS and sludge pH played a crucial role in sludge dewaterability.