Pacidamycins are a family of uridyl peptide antibiotics that inhibit the translocase MraY, an essential enzyme in bacterial cell wall biosynthesis that to date has not been clinically targeted. The pacidamycin structural skeleton contains a doubly inverted peptidyl chain with a β-peptide and a ureido linkage as well as a 3'-deoxyuridine nucleoside attached to DABA(3) of the peptidyl chain via an enamide linkage. Although the biosynthetic gene cluster for pacidamycins was identified recently, the assembly line of this group of peptidyl nucleoside antibiotics remained poorly understood because of the highly dissociated nature of the encoded nonribosomal peptide synthetase (NRPS) domains and modules. This work has identified a minimum set of enzymes needed for generation of the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation (T) domain (PacH) as a key carrier component in the peptidyl chain assembly as well as a freestanding condensation (C) domain (PacI) catalyzing the release of the assembled peptide by a nucleoside moiety. On the basis of the substrate promiscuity of this enzymatic assembly line, several pacidamycin analogues were produced using in vitro total biosynthesis.