Brassinosteroids (BRs) are widely used to mitigate the adverse effects of drought stress in plants. However, their varying effect between concentrations and soybean varieties is not well understood, which prevents their large-scale application. This study aimed to assess the effects of BRs on the physiological traits, water status and chlorophyll a fluorescence in soybean plants under drought stress. Two soybean varieties were studied, namely MONSOY8644 and FTR4288. The seeds were treated with five concentrations of 24-epibrassinolide-EBL (0.00, 0.05, 0.10, 0.15 and 0.20 mu M), under well-watered conditions (WW) and drought stress (DS) with complete irrigation suspension for seven days. Drought stress reduced relative water content, leaf succulence, SPAD index, soluble carbohydrates, and photochemical efficiency in plants, which led to cell membrane damage, identified by increased electrolyte leakage in both soybean varieties. These negative effects were more pronounced in the FTR4288 variety without EBL. However, concentration-dependent drought stress mitigation by EBL was evident in reduced electrolyte leakage and leaf temperature and increased proline content, which contributed to the osmotic adjustment of plants, improving their water status and alleviating damage to the photosynthetic apparatus. Our results indicate that adequate 24-epibrassinolide concentrations can be used as a strategy to mitigate the negative effects of drought stress in soybean plants, improving physiological traits. Seed treatment with 0.05 or 0.10 mu M of EBL is suggested as a useful method for alleviating drought stress in soybean and can therefore help farmers to grow the crop in areas with limited water resources.