The aim of the study was to assess the microbiological activity and clinical efficacy of colistin and rifampin combination against multidrug-resistant (MDR) Pseudomonas aeruginosa infections. The antimicrobial activity of the colistin/rifampin combination was evaluated using the checkerboard and time-kill curve methods against different MDR P. aeruginosa strains. The combination of rifampin and colistin resulted fully (1 strain) or partially (5 strains) synergistic for 6/7 strains and minimum inhibitory concentrations (MICs) in combination were reduced to easily obtainable therapeutic levels. The time-kill curves showed that the combination was bactericidal against the strains tested. The clinical efficacy of the combination was tested in four patients with difficult-to treat infections (sepsis or pneumonia) caused by MDR P. aeruginosa. All infections were successfully treated. Our microbiological and clinical observations suggest that the addition of rifampin to colistin may result in a synergistic bactericidal combination that may be useful in patients with infections caused by MDR P. aeruginosa which are difficult to cure.