With the increasing recognition of biofilms in human disease, the development of novel antimicrobial therapies is of critical importance. For example, in patients with cystic fibrosis (CF), the acquisition of host-adapted, chronic Pseudomonas aeruginosa infection is associated with a decline in lung function and increased mortality. Our objective was to test the in vitro efficacy of a membrane-active antimicrobial peptide we designed, termed 6K-F17 (sequence: KKKKKK-AAFAAWAAFAA-NH2), against multidrug resistant P. aeruginosa biofilms. This peptide displays high antimicrobial activity against a range of pathogenic bacteria, yet is non-hemolytic to human erythrocytes and non-toxic to human bronchial epithelial cells. In the present work, P. aeruginosa strain PAO1, and four multidrug resistant (MDR) isolates from chronically infected CF individuals, were grown as 48-hour biofilms in a static biofilm slide chamber model. These biofilms were then exposed to varying concentrations of 6K-F17 alone, or in the presence of tobramycin, prior to confocal imaging. Biofilm biovolume and viability were assessed. 6K-F17 was able to kill biofilms - even in the presence of sputum - and greatly reduce biofilm biovolume in PAO1 and MDR isolates. Strikingly, when used in conjunction with tobramycin, low doses of 6K-F17 significantly potentiated tobramycin killing, leading to biofilm destruction.