Due to the antimicrobial activity of flavonoids, it has been suggested that they may provide a possible alternative to antibiotics to stimulate productivity and reduce the environmental load of ruminant agriculture. We hypothesised that an extract of liquorice, rich in prenylated isoflavonoids and particularly glabridin, might potentially improve the efficiency of nitrogen utilisation and reduce methane production in the rumen. When added to a long-term rumen simulating fermentor (RUSITEC), liquorice extract at 1 g L−1 decreased ammonia production (−51%; P < 0.001) without affecting the overall fermentation process. When added at 2 g L−1, decreases in not only ammonia production (−77%; P < 0.001), but also methane (−27%; P = 0.039) and total VFA production (−15%; P = 0.003) were observed. These effects in fermentation were probably related to a decrease in protozoa numbers, a less diverse bacteria population as well as changes in the structure of both the bacterial and archaeal communities. The inclusion of an isoflavonoid-rich extract from liquorice in the diet may potentially improve the efficiency of the feed utilisation by ruminants.