The presence of the synthetic estrogen 17α-ethinylestradiol (EE2) in the environment is of increasing concern due to the endocrine disruption of aquatic organisms. Incomplete removal from wastewater (WW) is one of the main sources of EE2 in aquatic ecosystems, thus improving processes like biological WW treatment/activated sludge (AS) is becoming significantly important. There are opposing results regarding EE2 biodegradability by AS; one discrepancy is the efficacy of heterotrophic bacteria. This research demonstrated the ability of heterotrophs commonly present in AS (B. subtilis, P. aeruginosa, P. putida, R. equi, R. erythropolis, R. rhodochrous, R. zopfii) to remove EE2. R. rhodochrous was the most successful with no detectable EE2 after 48 h; the other bacteria achieved 21%-61% EE2 removal. No additive or synergistic effects were observed due to the combination of the bacterial cultures with maximum EE2 removals of 43% after 300 h.