Waste activated sludge is slower to biodegrade under anaerobic conditions than is primary sludge due to the glycan strands present in microbial cell walls. The use of pre-treatments may help to disrupt cell membranes and improve waste activated sludge biodegradability. In the present study, the effect of ultrasound, low-temperature thermal and alkali pre-treatments on the rheology, hygienization and biodegradability of waste activated sludge was evaluated. The optimum condition of each pre-treatment was selected based on rheological criteria (reduction of steady state viscosity) and hygienization levels (reduction of Escherichia coli, somatic coliphages and spores of sulfite-reducing clostridia). The three pre-treatments were able to reduce the viscosity of the sludge, and this reduction was greater with increasing treatment intensity. However, only the alkali and thermal conditioning allowed the hygienization of the sludge, whereas the ultrasonication did not exhibit any notorious effect on microbial indicators populations. The selected optimum conditions were as follows: 27,000 kJ/kg TS for the ultrasound, 80 °C during 15 min for the thermal and 157 g NaOH/kg TS for the alkali. Afterward, the specific methane production was evaluated through biomethane potential tests at the specified optimum conditions. The alkali pre-treatment exhibited the greatest methane production increase (34%) followed by the ultrasonication (13%), whereas the thermal pre-treatment presented a methane potential similar to the untreated sludge. Finally, an assessment of the different treatment scenarios was conducted considering the results together with an energy balance, which revealed that the ultrasound and alkali treatments entailed higher costs.