Rapeseed, the second-most-important vegetable oil source, is cultivated in various areas of India where both groundwater and soil are contaminated with fluoride (F–). Furthermore, the frequent use of F– contaminated groundwater for irrigation leads to accumulation of F– in surface and sub-surface soil. The study aims to compare the morphological and biochemical changes in Brassica juncea L., the variations in its fatty acids (FAs) composition and oil yield, under two regimes of F– contaminated soils: (i) pre-contaminated soil (Tr) and (ii) irrigation with F– contaminated water (Ir). The level of F– (µg g−1) in the plant tissues (root, leaf, and grain) was significantly higher in Ir_10 (18.3, 14.7, and 2.8, respectively) than in Tr_10 (4.3, 2.6, and 0.77, respectively), while the oil yield was significantly lower with Ir_10 (19.5%) than with Tr_10 (44.9%). The phytoremediation potential of F– by Brassica juncea L. is greater in Tr regime than in the Ir regime. The erucic acid content (%), which is detrimental to cardiac health, increased to 67.37% (Ir_10) and 58.3% (Tr_10) from 57.73% (control). Thus, the present study shows that irrigation with F– contaminated water results in greater toxicity and accumulation in plants and is not safe for human health.