Anaerobic digestion (AD) is a technology for recovering chemical energy as methane from excess sludge/waste. Unfortunately, humic acids (HA) contained in excess sludge can have the effects of inhibiting the efficiency of energy conversion. Based on a batch experiment, the impact of HA on a semi-continuous AD process was sequentially investigated, with the impact on the associated enzymes and microorganisms being measured. The results of this semi-continuous experiment indicate that the inhibition of the microbial community increased with an increased HA:VSS ratio. Long-term cultivation did not result in the adaption of methane production to the presence of HA. Moreover, at HA:VSS = 20%, the strongest inhibition (74.3%) on energy conversion efficiency was observed in the semi-continuous experiment, which was two-fold higher than that recorded in the batch experiment. This is attributed to serious and irreversible inhibition of both acidogenic and methanogenic microorganisms, as well as the physical-chemical reactions between HA and the associated enzymes which, it was concluded, were the dominant mechanisms of inhibition in the batch experiment.