Vancomycin, a glycopeptide antibiotic often prescribed for staphylococcal infections (particularly methicillin-resistant strains), is isolated from fermentation broth as vancomycin B (formulated as vancomycin hydrochloride). A monodechlorovancomycin with a retention time similar to vancomycin B in USP and EP HPLC assays is formed during vancomycin fermentation. We sought to isolate this compound for use as an HPLC standard and test its antibacterial activity against vancomycin B. Vancomycin contains two chlorine atoms, giving two possible monodechloro products: Monodechlorovancomycin 1 was chemically synthesized by reducing vancomycin B, while Monodechlorovancomycin 2 was isolated from fermentation broth. HPLC and NMR confirmed the two compounds are distinct, with Monodechlorovancomycin 2 as the fermentation concomitant (Monodechlorovancomycin 1 not detected in fermentation; didechlorovancomycin found). Herein, we report the synthesis, isolation, and antibacterial activities of the two monodechlorovancomycins. Both had nearly identical UV spectra (secondary absorption maximum ~290nm) and mass spectra indicating one chlorine atom. Antibacterial tests (broth microdilution per NCCLS) showed they shared vancomycin B’s spectrum (inhibiting Gram-positive staphylococci, vancomycin-susceptible enterococci, Streptococcus pneumoniae) but slightly reduced activity (~70% of vancomycin B). They had no activity against Gram-negative pathogens (except weak activity against Moraxella catarrhalis), Candida albicans, or vancomycin-resistant enterococci. Teicoplanin was active against inducible VanB strains.