This research focuses on assessing the levels, distribution, and ecological risks of polycyclic aromatic hydrocarbons (PAHs) in sediments and water from the Klip River, Johannesburg, South Africa. These persistent organic pollutants classified by the United States Environmental Protection Agency (US EPA) as carcinogenic, mutagenic, and teratogenic, posing serious risks towards aquatic animals, human health and the environment. The study employed liquid-liquid extraction (LLE) for water samples and microwave-assisted extraction (MAE) for sediments, with subsequent analysis by gas chromatography-flame ionization detection (GC-FID). Method efficiencies were verified using spiked water samples and certified reference material (CRM-104). Ecotoxicological effects were evaluated using the zebrafish embryo development test (ZFET), which revealed that sediment samples posed significantly higher toxicity than water samples, causing low hatch rates, high mortality, and developmental malformations. These findings highlight the urgent need for monitoring and remediation to protect the environment, aquatic life and to reduce the potential cancer risk and other health impacts for communities relying on this water system.