Plant fatty acids (FAs) are critical components of lipids and play an important role in coping with pollution-induced stress. However, the relationship between the fluctuating changes of FAs and the toxic effects of pollutants is not clear. Here, we analyzed and identified 19 FAs, namely 14 medium and long chain fatty acids (MLCFAs) and 5 very long chain fatty acids (VLCFAs). First, a positive correlation between plant biomass and LCFA content was observed. Changes in unsaturation were inversely related to cell membrane permeability, which serves as an indicator of the toxic effects. In particular, the use of herbicides led to a reduction in total FA content, but caused a significant increase in free fatty acids (FFAs), which facilitate oxidative stress. In addition, supplementation with exogenous FAs, particularly linoleic and alpha-linolenic acids, effectively alleviated the toxic inhibition. (R)-dichlorprop causes abnormal FA metabolism that can be reversed by ferrostatin-1, a ferroptosis inhibitor. Under (R)-dichlorprop exposure, the balance of FA unsaturation in plants is disrupted by inhibition of FA desaturase activity, ultimately leading to ferroptosis and disruption of cell membrane integrity. This study aims to enhance the understanding of the ecotoxic effects of herbicides by examining changes in FAs. The findings will provide a scientific basis for controlling environmental risks associated with hazardous substances.