The marine bacterium Pseudoalteromonas SANK73390 produces thiomarinols A–G, hybrid antibiotics that link a pyrrothine moiety to analogues of the clinical antibiotic mupirocin, both of which exhibit potent activity against MRSA. The thiomarinol (tml) biosynthetic gene cluster is located on a 97 kb plasmid and includes trans-AT polyketide synthases (PKSs), a nonribosomal peptide synthetase (NRPS), and tailoring enzymes. We report the full metabolic profiles and characterization of previously undetected metabolites in wild-type (WT) and mutant (ΔPKS, ΔNRPS, ΔtmlU) strains of Pseudoalteromonas SANK73390, including an inframe deletion mutant (ΔtmlU) of the TmlU amide synthetase. Mutasynthesis experiments using selected biosynthetic substrates in ΔPKS and ΔNRPS mutants yielded new thiomarinol analogues with altered activity against mupirocin-resistant MRSA. Antimicrobial testing confirmed these analogues have good anti-MRSA activity. Combined with our prior identification of the tml gene cluster, this study provides the first evidence that engineered Pseudoalteromonas SANK73390 strains enable the rational production of new anti-MRSA compounds via mutation and mutasynthesis, representing significant potential for developing clinically relevant anti-MRSA agents.