People with cystic fibrosis experience re-occurring polymicrobial pulmonary infections that are a leading cause of mortality. Among the infecting organisms is the Gram-negative Burkholderia cenocepacia, which causes a rapidly progressing form of necrotizing pneumonia and bacteremia known as “cepacia syndrome”, with few treatment options due to high intrinsic antibiotic resistance. The application of next-generation sequencing has powered recent genome-wide explorations into antibiotic resistance. Here, we lay the foundation for such an exploration into the epidemic clinical isolate B. cenocepacia K56-2 with an in-depth survey into its resistance arsenal. We have characterized growth dose-response curves for a panel of 87 antimicrobials from over 30 diverse classes, including clinically relevant antibiotics. Despite many not causing full growth inhibition, we observed important differences and similarities in and among structural classes. For example, tetracyclines and quinolones were up to two orders of magnitude more potent than aminoglycosides and cationic peptides. Within the cephalosporins, while ceftazidime by itself has poor activity, the related siderophore conjugate, cefiderocol, possessed a 512-fold lower MIC. Furthermore, cross-reference to the Comprehensive Antibiotic Resistance Database (CARD 2020) explains many of the observed trends in antimicrobial activity. K56-2 putatively encodes over 250 unique known resistance genes, including carbapenemases, broad-spectrum efflux pumps, and outer membrane modification systems. Furthermore, we have constructed and validated a high-density barcoded transposon mutagenesis scheme to quantitatively profile genomic contributions to antimicrobial resistance. At sub-inhibitory concentrations of antibiotics, hypersusceptible mutants will be selectively killed, which can be monitored by next-generation sequencing and enumeration of unique DNA barcodes in each mutant. Current efforts are focused on cefiderocol, ceftazidime-avibactam, meropenem, and aztreonam; however, our high-throughput platform, will allow us to profile all 87 antimicrobials in the panel. We expect these studies to yield valuable insight into novel therapeutic avenues for treating infections caused by B. cenocepacia and related bacteria.