A rapid and sensitive analytical method for the determination of oxolinic acid and flumequine in aqueous samples based on dispersive liquid-liquid microextraction using ionic liquids (ILs) was developed. Based on the structural properties of the antibiotic agents studied, two ILs with different functionalities were required: ethyl-dimethyl-(2-methoxyethyl) ammonium tris(pentafluoroethyl)trifluorophosphate ([MOEDEA][FAP]) for extraction and 1-butyl-3- methylimidazolium tetrafluoroborate ([BMIM][BF4]) to adjust the polarity of the medium. The significant experimental factors involved in DLLME were identified and optimized using the experimental design methodology. A Plackett-Burman design was initially used for screening, and a central composite design was used for optimization. The optimized method exhibited good precision, with relative standard deviation values of less than 5 % and limits of detections on the order of 0.1 and 0.3 ng mL-1 for the two drugs. The enrichment factors for both antibiotics were 13–33 fold. The proposed method was applied to the analysis of the two antibiotic in spiked surface, river, and wastewater samples.