A direct bioautography has been used for the simultaneous determination of four estrogens [estrone (E1), 17β-estradiol (E2), estriol (E3), and 17α-ethinylestradiol (EE2)] and two xenoestrogens [bisphenol A (BPA) and 4-n-nonyl-phenol (NP)] in surface water and wastewater samples from a sewage treatment plant. After either direct application or a liquid-liquid extraction of the water samples, the qualitative and quantitative detection of estrogen-effective compounds was performed with a planar yeast estrogen screen. The limits of detection were different for each compound, due to the specific receptor binding of individual (xeno)estrogens (1 ng/L to 15 μg/L). The mean recovery rate for all six substances at this ultratrace level was 88% [mean percent relative standard deviation (%RSD) of 17%, n = 3]. Over the whole procedure, precisions of three estrogens discovered in a wastewater sample were below 17%, n = 3. The identification of the detected bioactive compounds was performed by high-performance thin-layer chromatography-electrospray ionization mass spectrometry (HPTLC-ESI-MS) via the elution-head-based TLC-MS Interface. Whereas the estrogens E1 and E2 could always be detected in the influent of the treatment plant, E3 was detected occasionally. The concentrations of E1 and E2 ranged from 3 to 50 ng/L, and for E3 from 98 to 210 ng/L. EE2, BPA, and NP could not be detected at the given LOD. In every second surface water sample, E1 and E2 were detected, but not E3, EE2, BPA, and NP.