The rapid increase in antibiotic resistance has prompted the discovery of drugs that reduce antibiotic resistance or new drugs that are an alternative to antibiotics. Plant extracts have health benefits and may also exhibit antibacterial and antibiofilm activities against pathogens. This study determined the antibacterial and antibiofilm effects of α-humulene extracted from plants against enterotoxigenic Bacteroides fragilis (ETBF), which causes inflammatory bowel disease. The minimum inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) of α-humulene for B. fragilis were 2 μg/mL, and the biofilm eradication concentration (Pumbwe et al.) was in the range of 8-32 μg/mL. The XTT reduction assay confirmed that the cellular metabolic activity in biofilm rarely occurred at 8-16 μg/mL concentration. In addition, the biofilm inhibition by α-humulene was also detected via confocal laser scanning microcopy. Quantitative real-time polymerase chain reaction was also used to investigate the effect of α-humulene on the expression of resistance-nodulation-cell division (RND)-type multidrug efflux pump (bmeB1 and bmeB3) genes. As a result of qPCR, α-humulene significantly reduced the expression of bmeB1 and bmeB3 genes. This study demonstrates the potential therapeutic application of α-humulene to inhibit the growth of B. fragilis cells and biofilms, and contribute to the expansion of knowledge about biofilm medicine.