Standard disc diffusion antimicrobial susceptibility testing (C+S) on individual Pseudomonas aeruginosa colonial morphotypes cultured from cystic fibrosis (CF) sputum has questionable clinical relevance. Direct sputum sensitivity testing (DSST) is a whole-sputum susceptibility test that removes bias associated with selecting individual colonial morphotypes. We sought to determine whether, in principle, the results from DSST support the possibility of improved clinical relevance compared with C+S. Individual (DSSTi) and combination (DSST) susceptibility to gentamicin, tobramycin, ceftazidime and meropenem were determined on 130 sputum samples referred from CF subjects with antibiotic-resistant chronic Gram-negative endobronchial infection. DSSTi and concurrent C+S were compared for categorical susceptibility, synergistic combinations were evaluated and the combination DSST efficacy index (DEI) calculated. Meropenem and tobramycin were the most active individual antibiotics by DSSTi on 89 P. aeruginosa-predominant samples, with 62 % of samples sensitive to each. C+S and DSSTi showed poor agreement (κ ranging from 0.02 to 0.6), discordance ranging from 20 % (meropenem) to 49 % (tobramycin), with DSSTi demonstrating both increased susceptibility and increased resistance. The combination that most frequently had the highest DEI was tobramycin + meropenem, occurring in 76 % of samples. DSSTi appears to be reproducible, yields different antimicrobial susceptibility results from C+S without simply identifying the most resistant isolates and DSST identifies the most effective in vitro antibiotic combinations, providing preliminary proof of concept of the potentially improved clinical relevance of whole-sputum testing. Future studies will determine whether these potential theoretical advantages translate into clinical benefits.