A wide range of synthetic chemicals and their metabolites present in the environment can antagonise the receptor activity of androgen hormones present in wildlife and humans. With increasing global production of new synthetic chemicals, little is known about their environmental fate, health consequences and end-points. This study was conducted to identify and characterise chemicals with anti-androgenic activity present in wastewater influent and effluent. This study was undertaken by applying a combination of biological and analytical chemistry techniques involving Solid Phase Extraction (SPE), High Performance Liquid Chromatography (HPLC) and an in vitro steroid receptor assay for profiling and characterising extracts of grab influent and effluent wastewater samples using a toxicity identification and evaluation (TIE) procedure. Initial work revealed variable recoveries of anti-androgenic activity from SPE of wastewaters. Therefore SPE methodology to screen wastewater samples was developed using a mixture of selected compounds which possess a range of polarities (log Kow). Their recoveries from SPE were measured by HPLC protocol and ranged from 95- 100%. The mean±SD and % RSD values of the analysed wastewater replicates were 3.20±0.03 mgFeq/L and 0.78% for influent and 0.22±0.01 mgFeq/L and 3.80% for effluent samples. The recoveries of wastewater extracts after fractionation were between 78.6% and 99.6%. Fractions containing anti-androgenic activity were analysed by Gas Chromatography Mass Spectrometry (GC-MS). A number of household chemicals were detected in both influent and effluent wastewater fractions that contained antiandrogenic activity. These included the anti-bacterial agents- triclosan, chlorophene, dichlorophene, chloroxylenol, the musk fragrance galaxolide, the flame retardantstris( 1-chloro-2-propyl)phosphate (TCPP) and tris(2-butoxyethyl)phosphate (TBEP), polymer plasticizer n-butylbenzenesulfonamide (NBBSA) and bisphenol A (BPA) which is a chemical associated with the polycarbonate usage. The anti-androgenic potency of pure contaminants compared with that of flutamide ranged from 0.04 (TCPP) to 13.40 (chlorophene). Anti-androgenic activity of 1.69 and 2.00% was recovered from the fractions of the effluent and influent samples respectively indicating that AA of about 98% are yet to be recovered. This work reveals for the first time that over 12 contaminants contribute to the total anti-androgenic activity present in wastewater effluent and that a number of compounds commonly used in household products (such as chlorophene, triclosan and NBBSA) are predominant anti-androgens in wastewater effluents.