The aim of the study was to develop self-emulsifying drug delivery systems (SEDDS) providing a synchronized delivery of an antibiotic drug and a complexing agent for enhanced antimicrobial efficacy. Polymyxin B (PMB) was ion paired with agaric acid (AA) serving as complexing agent and lipophilic counter ion and incorporated into SEDDS. Resazurin assay was performed to assess toxicity of SEDDS having been diluted with minimum essential medium. SEDDS were diluted 1:10 in 0.01 M NaOH and evaluated for their ability to bind Ca2+ and Mg2+ as well as for enhanced antimicrobial activity using E. coli as model germ. Resazurin assay revealed that >80% of Caco-2 cells remain viable up to a SEDDS concentration of 0.5% (v/v) within 4 h. SEDDS showed chelating properties by binding 18.7 +/- 0.7 g of Ca2+ and 12.0 +/- 0.6 g of Mg2+ per mol of AA accumulating to a high extent on the surface of the oily droplets. SEDDS containing the PMB-AA complex and AA exhibited 36- and 13- fold enhanced antimicrobial activity compared to SEDDS containing just AA or the PMB-AA complex, respectively. The combination of PMB with a complexing agent serving also as lipophilic counter ion for hydrophobic ion pairing seems to be a promising strategy to enhance the antimicrobial activity of this antibiotic.