The bacterial genus Enterococcus has been reported as commensal bacterium inhabiting gastrointestinal tracts of humans. Members of Enterococcus can be used as a probiotic or act as opportunistic pathogen causing urinary tract infection. However, bioactive metabolites produced by these bacteria are still largely unknown. Nine new phenylethylamine-containing imidazolium, enterozolium A–I (1­­­­–9) were isolated from E. faecalis KCTC 5191 along with five known metabolites (10–14). Chemical structures of the new compounds (1–9) were elucidated by spectroscopic data analysis, including NMR (¹H and ¹³C NMR, ¹H-¹H COSY, HSQC, HMBC) and HR-TOF-MS data. Enterozolium F exhibited potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Bacillus subtilis, with MIC values ranging from 1.3–2.6 µg/mL. The biosynthetic pathway toward enterozolium A–I (1–9) was investigated using biomimetic total synthesis, isotope labeling experiments and a biosynthetic genes knockout method.