Environmental pollution due to di-ethyl phthalate (DEP) is one of the major concerns all over the world. This is probably the first study regarding the biodegradation of DEP by newly isolated Pseudomonas juntendi strain CCNU-SK1, P. putida strain CCNU-SK2 and P. nitritireducens strain CCNU-SK3. Bacterial strains were isolated by morphological, biochemical, physiological and 16S rDNA gene sequencing techniques. The biodegradation of DEP was quantified, and degradation kinetics were studied by using high-performance liquid chromatography. The optimum growth of bacteria for strain CCNU-SK1 was obtained at 28 °C, pH 8, DEP (500 mg/L), NaCl (1.5%), half-life (10.19 h); in case of strain CCNU-SK2: 33 °C, pH 7, DEP (400 mg/L), NaCl (1.5%), half-life (13.86 h) and for strain CCNU-SK3: 33 °C, pH 7, DEP (500 mg/L), NaCl (1.75%), half-life (12.6 h). Strain CCNU-SK1 with lowest half-life period was the most efficient bacteria for biodegradation of DEP compared to other isolated strains. More than 99% of all DEP were biodegraded within the 4 days and the complete utilization of (50 mg/L) DEP occurred within 24 h by all strains. All of them were capable of metabolizing not only DEP but also other phthalate esters (PAEs) such as DMP (Di-methyl phthalate), DBP (Di-butyl phthalate), DOP (Di-octyl phthalate), BBP (Benzyl butyl phthalate), DEHP (Di-2-ethylhexyl phthalate) and PA (Phthalic acid), making them the promising agents for the bioremediation in the PAEs contaminated sites.