BACKGROUND Treatment of infective endocarditis secondary to Pseudomonas aeruginosa can be challenging because of this organism's ability to acquire antimicrobial resistance over time. METHODS We describe a patient with native aortic valve infective endocarditis due to P. aeruginosa who developed progressive multi-drug resistance while on therapy. The resistance mechanisms were characterized using whole-genome sequencing. RESULTS We identified two mutations in subsequent isolates (dacB and OprD) that conferred resistance to anti-pseudomonal penicillins, cephalosporins, and carbapenems. The patient was treated with combination high-dose continuous infusion meropenem and ciprofloxacin therapy, in addition to bioprosthetic aortic valve replacement and repair of ventricular septal wall defect. Antibiotics were continued for 6 weeks post-cardiac surgery and the patient remains infection free 18 months post-completion of antibiotic therapy. CONCLUSIONS Clinicians should be aware of the ability of P. aeruginosa to acquire resistance mechanisms in response to selective antibiotic pressures in high-inoculum infections such as infective endocarditis. The mutations identified in this case report correlated well with the evolving antimicrobial resistance profile observed.