The Yamuna River water-induced phytotoxicity was investigated in mustard seeds (Brassica campestris L.). Phytotoxicity assays were conducted on water samples from the Yamuna River in Delhi to assess the water quality at the Wazirabad barrage (upstream), ITO barrage (midstream), and Okhla barrage (downstream) sites. Two samples were collected from the Wazirabad barrage, another from the ITO barrage, and a third from the Okhla barrage. When compared to plants grown under normal conditions, seedlings exposed to 50% sample concentration showed significant increases in root and shoot length and biomass by 5% and 25%, respectively. After 5 days of treatment, there was a reduction in germination. Mustard seedlings that were exposed to water samples with concentrations ranging from 5 to 50% v/v showed a significant decrease in germination index compared to the control plants (0%), indicating water-induced phytotoxicity. Rapid industrialization and development have led to river water pollution, posing a serious concern. This form of pollution is often irreversible. In Delhi-NCR, industrial and domestic waste have become a major threat to the Yamuna River, which is a crucial water source. The Central Pollution Control Board (CPCB) has labelled the river as "unfit for drinking" and "unsafe for bathing" due to high pollutant levels. Recent studies have indicated a deterioration in the water quality of the Yamuna River, which has been found to have harmful effects on various plant models.