Aims: To select and evaluate an appropriate outer membrane (OM) permeabilizer to use in combination with the highly muralytic bacteriophage endolysin EL188 to inactivate (multi-resistant) Pseudomonas aeruginosa. Methods and Results: We tested the combination of endolysin EL188 and several OM permeabilizing compounds on three selected Ps. aeruginosa strains with varying antibiotic resistance. We analysed OM permeabilization using the hydrophobic probe N-phenylnaphtylamine and a recombinant fusion protein of a peptidoglycan binding domain and green fluorescent protein on the one hand and cell lysis assays on the other hand. Antibacterial assays showed that incubation of 106Ps. aeruginosa cells ml-1 in presence of 10 mmol l-1 ethylene diamine tetraacetic acid disodium salt dihydrate (EDTA) and 50 mu g ml-1 endolysin EL188 led to a strain-dependent inactivation between 3 center dot 01 +/- 0 center dot 17 and 4 center dot 27 +/- 0 center dot 11 log units in 30 min. Increasing the EL188 concentration to 250 mu g ml-1 further increased the inactivation of the most antibiotic resistant strain Br667 (4 center dot 07 +/- 0 center dot 09 log units). Conclusions: Ethylene diamine tetraacetic acid disodium salt dihydrate was selected as the most suitable component to combine with EL188 in order to reduce Ps. aeruginosa with up to 4 log units in a time interval of 30 min. Significance and Impact of the Study: This in vitro study demonstrates that the application range of bacteriophage encoded endolysins as 'enzybiotics' must not be limited to gram-positive pathogens.