17β-estradiol (E2) is the natural estrogen with the most significant potential for endocrine disruption in the biota of aquatic ecosystems at trace concentrations. It is, therefore, essential to study treatments for water polluted with E2 that would guarantee its complete elimination and mineralization. Denitrification is a biological process shown to have the capacity to completely biodegrade drugs, such as ampicillin. This work is aimed to evaluate the biotransformation of 17β-estradiol by employing a denitrifying sludge. The assays performed were: (I) abiotic with 3.5 mg E2-C L−1 and (II) denitrifying with 10 mg CH3COO−-C L−1 as the reference, 10 mg E2-C L−1 as the sole electron donor, and a mixture of (mg L−1) 10 E2-C with 10 CH3COO−-C at C N−1 of 1.1. The E2-C and NO3−-N consumption efficiencies were greater than 99%, and HCO3−-C and N2 production yields were close to 1 in all assays. The denitrifying sludge could biodegrade up to 10 mg E2-C L−1 as the sole electron donor and when mixed with 10 mg CH3COO−-C L−1. No intermediate metabolites were generated from the process.