Estrogen in streams threatens aquatic animals, especially where wastewater treatment plant (WWTP) effluent contributes to baseflow. We investigated total estrogen (E1+E2+E3) as estradiol equivalent (E2) and ethynylestradiol (EE2) concentration in Cibolo Creek (Cibolo), a groundwater-fed stream near San Antonio, TX, receiving effluent via two WWTP. We collected water samples bi-monthly from late spring to early fall 2018 in Cibolo and WWTP effluent, and used ELISA analysis and discharge measurements to determine concentrations and loads of estrogens. We measured several environmental variables to investigate what factors influenced estrogen concentrations in Cibolo downstream from WWTP inputs. Mean concentrations of WWTP effluent (E2, 41.43 +/- 15.48; EE2, 11.40 +/- 2.07 ng L-1) were higher compared with concentrations in Cibolo, both downstream (E2, 30.09 +/- 25.85; EE2, 6.33 +/- 1.92 ng L-1) and upstream (E2, 12.91 +/- 11.12; EE2, 4.5 +/- 1.38 ng L-1) of WWTP inputs. Both E2 and EE2 concentrations decreased downstream from WWTP inputs, a section of stream without large quantities of fine sediments for sorption, indicating potential dilution or chemical and biological degradation. Effluent into Cibolo via the first, and older, WWTP contributed the most estrogen load in Cibolo. Median concentrations of E2 and EE2 were 19 and 5 ng L-1, respectively, downstream of WWTP inputs, concentrations known to affect reproductive processes of aquatic biota and impair human health. Results suggest estrogens may pose a risk to aquatic ecosystems wherever WWTP effluent comprises a majority of baseflow, though further studies are required in this stream to verify biological impacts.