Twelve bacteria were isolated from wastewater for the degradation of 17β-estradiol (strain K1, K2, K3, K4, L5, L6, L7, L8, M9, M10, M11 and M12) by enrichment method. Initial screening revealed maximum degradation (78 ± 1.1%, and 86 ± 4.2%) of 17β-estradiol by strains L7, and M10, respectively. The production of manganese peroxidase by bacteria gets involved in the degradation of various contaminants. Almost all strains isolated from the enriched medium produced manganese peroxidase. Based on initial screening on 17β-estradiol degradation and manganese peroxidase production, two bacteria (Flavobacterium longum L7 and Pseudomonas aeruginosa M10) were selected to analyze 17β-estradiol degradation in co-culture system. The co-cultured strains could rapidly remove 17β-estradiol (91.3%) from the culture medium at 10 mg/L 17β-estradiol initial concentration. Moreover, degradation of 17β-estradiol was only 82% and 75% when the strains L7 and M10 were treated individually. Initial 17β-estradiol concentration was 10 mg/L and 0.3% inoculum were optimum and achieved >90% degradation. Alkaline pH was optimum (pH 7.5) and reached 98.4 ± 7.5% degradation in co-culture. Glucose and trehalose improved the growth and degradation of 17β-estradiol. The organic nitrogen sources such as, yeast extract and peptone improved the removal of 17β-estradiol. The selected bacterial strains were resistant against penicillin, erythromycin, oxytetracycline and tetracycline. The present findings demonstrated the application of F. longum L7 and P. aeruginosa M10 co-culture for the removal of steroid hormones in the wastewater.