Chromium (Cr) contamination in agricultural soils, largely due to industrial activities, poses a significant threat to plant growth and productivity. This study examines the effects of Cr stress at concentrations of 100 and 200 mg of K2Cr2O7 per kg soil on rapeseed (Brassica napus) roots and evaluates the mitigating potential of biochar. Biochar, produced through both slow and fast pyrolysis and applied at 30 g per kg soil, was investigated for its ability to neutralize Cr toxicity. Our findings indicate that Cr stress significantly decreased the growth and physiological functions of rapeseed roots. However, biochar application improved soil pH, cation exchange capacity, and the uptake of essential nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium. Additionally, biochar enhanced the production of osmotic regulators like glycine betaine and soluble proteins, as well as indole acetic acid, promoting better root growth and water uptake under Cr stress. Notably, biochar reduced Cr availability and absorption in rapeseed roots, leading to lower levels of stress-related hormones such as abscisic acid, salicylic acid, and jasmonic acid. Among the biochars tested, slow pyrolysis biochar was more effective than fast pyrolysis biochar in mitigating Cr toxicity. These results highlight the potential of slow pyrolysis biochar as a sustainable strategy to alleviate Cr pollution and enhance plant resilience in contaminated soils.