The multinuclear non-heme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, post-translationally modified peptide natural products (RiPPs). The few MNIOs characterized to date catalyze a breadth of unusual oxidations, primarily on cysteine residues, including oxazolone and thioamide formation, carbon excision, amino acid cleavage, and macro- and heterocycle formation. Recently, a secreted virulence factor termed HvfA from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO, HvfB. Spectroscopic characterization of HvfB shows that it is isolated with a mixture of di- and triferric cluster. In the presence of HvfC (a RiPP recognition element (RRE)-containing partner protein), HvfB is readily reduced to form an S = ½ signal indicative of a coupled Fe(II)/Fe(III) species, suggesting the mixed-valent state is key for activity. We demonstrate that HvfB works together with HvfC to perform six post-translational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR show that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor uses these modified residues to coordinate Cu(I) ions. Considering the necessity of HvfA for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection.