Ingestion of pyrrolizidine alkaloids, naturally occurring plant toxins, causes illness and death in a number of animal species. Senecio jacobaea pyrrolizidine alkaloids cause significant economic losses due to livestock poisoning, particularly in the Pacific Northwest. Some sheep are resistant to pyrrolizidine alkaloid poisoning, because ovine ruminal biotransformation detoxifies free pyrrolizidine alkaloids in digesta. Antibacterial agents modify ruminal fermentation. Pretreatment with antibacterial agents may account for some animal variability in resistance to pyrrolizidine alkaloid toxicosis, and antibacterial agents can also be used for characterizing ruminal pyrrolizidine alkaloid-biotransforming microflora. The objective of this study was to evaluate the effects of antibacterial agents on biotransformation of a predominant S. jacobaea pyrrolizidine alkaloid, jacobine, in ovine ruminal contents. Ovine ruminal jacobine biotransformation was tested in vitro with 20 independent antibacterial agents. Low amounts of rifampin and erythromycin prevented jacobine biotransformation. Chlortetracycline, lasalocid, monensin, penicillin G, and tetracycline were slightly less effective at inhibiting jacobine biotransformation. Bacitracin, crystal violet, kanamycin, and neomycin were moderately inhibitory against jacobine biotransformation. Brilliant green, chloramphenicol, gramicidin, nalidixic acid, polymyxin B SO4, sodium azide, streptomycin, sulfisoxazole, and vancomycin had little to no effect on jacobine biotransformation. The antibiotics that were most effective at inhibiting biotransformation were those that are active against gram-positive bacteria. Therefore, gram-positive bacteria are most likely critical members of the jacobine-biotransforming consortia.