Abstract Background: In 2001, the U.S. Food and Drug Administration (FDA) convened to conduct a safety assessment of a plasticizer, di(2-ethylhexyl) phthalate (DEHP), released from polyvinyl chloride (PVC) medical devices. Hospitalized patients may be exposed to high concentrations of plasticizers, antioxidants, and chemical contaminants in PVC medical devices during blood transfusion or hemodialysis, thus, making them vulnerable to more potentially adverse effects of these chemicals than healthy people. At the same time, recently, the effect of endocrine-disrupting chemicals on hospitalized patients has attracted a great deal of attention. This study aims to investigate the harmful effects of estrogenic compounds in medical PVC tubing by two approaches of gas chromatography–mass spectrometry (GC–MS) and estrogen receptor (ER) binding assay. Methods: Residual plasticizers, antioxidants, and chemical contaminants in PVC tubing were subjected to GC–MS in the full-scan mode with an original library for plastic additives. Such residual compounds in PVC tubing were screened at very low concentrations (10 −2–10 5 nmol/l) to determine whether they competed with fluorescein-labeled estradiol for ER (α). Results: DEHP, 2-ethylhexanol, butylated hydroxytoluene, and 4-nonylphenol (NP) were detected in medical PVC tubing. In addition, only NP in PVC tubing was found to bind with ER. Conclusions: The current study proves that the main residual chemical for estrogenic effect was NP in medical PVC tubing.