Anoectochilus roxburghii(Wall.) Lindl. is a precious herb plant and its rhizome cultures can be used as an alternative plant material. To utilize rhizomes in practical production, this study investigated the antibacterial and anti-biofilm effects of the rhizome extract (RhE) from (A) roxburghii, and further clarified the antibacterial mechanism. Results showed that RhE inhibited the growth of all tested bacterial species (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis) and the highest inhibitory zone (18.6 mm) was found on (B) subtilis, which was 4.8 mm more than the extract (PE) from the field-grown plants of (A) roxburghii. The RhE treatment increased the leakage of the alkaline phosphatase, electrolyte, nucleic acid, and protein of (B) subtilis, indicating that the cell permeability was increased. Furthermore, RhE inhibited the respiration of B. subtilisand decreased the activity eof the key enzyme (glucose-6-phosphate dehydrogenase) in the hexose monophosphate (HMP) pathway, proving that RhE was involved in the HMP pathway. In addition, RhE inhibited bacterial biofilm formation, which was higher than PE; Among the four tested bacteria, RhE treatment had strong inhibitory effect on biofilm formation of P. aeruginosaand B. subtilis. The findings suggested that A. roxburghiirhizome cultures are the potential material in the production of antibacterial and anti-biofilm products.