Indoor dust serves as a potential sink for various synthetic chemicals used in our daily lives, while exposure to these anthropogenic contaminants via dust contact, ingestion, or inhalation may pose potential threats to human health. In this study, in vitro biological assays were used to investigate the endocrine disrupting activity and genotoxicity in dust samples collected from a university located in southern Taiwan. Contents of polycyclic aromatic hydrocarbons (PAHs) in indoor dust were also analysed by gas chromatography mass spectrometry. Our results showed that significant aryl hydrocarbon receptor (AhR) agonist, antiandrogenic, antithyroid hormonal, and genotoxic activities were found in dust samples. In particular, high AhR agonist activities were found in indoor dust collected from computer room and laboratory (16112 and 9686 ng benzo(a)pyrene equivalent/g dust dry weight), whereas AhR agonistic PAHs were responsible for only a small percentage of the bioassay-derived activities. Higher antiandrogenic and genotoxic activities were found in indoor dust from office and classroom, respectively, suggesting that contaminants varied in different indoor dust samples. After fractionating by high performance liquid chromatography, AhR agonist activities were detected in several fractions of indoor dust from computer room and laboratory, indicating the presence of unknown AhR agonist contaminants in these indoor dust samples. Further isolation and identification of novel AhR agonistic and antiandrogenic contaminants is necessary to protect the environment and human health.