The influence of the microbial activity on the strength of activated sludge flocs was studied in short term experiments (0–3 h). Increased floc strength was generally obtained when the aerobic microbial activity was stimulated by adding substrate. Deflocculation was observed when the aerobic microbial activity was inhibited by (i) anaerobic conditions; (ii) addition of the metabolic inhibitors azide and chloramphenicol; and (iii) reduction of the temperature to 4°C. Furthermore, addition of nitrate as electron acceptor under anaerobic conditions partly prevented deflocculation from taking place. These results strongly suggested that microorganisms using oxygen and/or nitrate as electron acceptors were important for maintaining the floc strength. The increase in turbidity under deflocculation was well correlated with the number of bacteria and concentration of protein, humic substances and carbohydrates in the supernatant. However, only approximately 1–2% of the total amount of sludge deflocculated, so the deflocculation could be understood as an erosion of small particles from the larger flocs. The extent of deflocculation under anaerobic conditions could be enhanced by stimulation of the anaerobic biological activity. When anaerobic conditions prevailed, a microbial iron reduction immediately started with iron reduction rates of 4–150 μmol/gVS·h. Subsequently, a decrease in floc strength was observed which could also be observed when the iron-reducing bacterium Shewanella alga BrY was added to the activated sludge. Furthermore, the importance of Fe(III) for the floc strength was illustrated by removal of Fe(III) from the sludge matrix by adding sulphide, which resulted in strong deflocculation. Thus, the deflocculation observed could be either directly due to lack of aerobic microbial activity or indirectly due to change in the local physico-chemical conditions mediated by anaerobic microbial activity.