Chromium (Cr) contamination of agricultural soils is a major threat to human and plant health worldwide and causes reduc- tions in plant growth and crop yields. 24-epibrassinolide (EBL) and nitric oxide (NO) have been shown to ameliorate the reductions in growth caused by the stresses induced by heavy metals; however, the interactions between EBL and NO on the alleviation of Cr-induced phytotoxicity have been poorly studied. Hence, this study was undertaken to examine any benefi- cial effects of EBL (0.01 μM) and NO (100 μM), applied alone or in combination, on the mitigation of stress induced by Cr (100 μM) in soybean seedlings. Although EBL and NO applied alone reduced the toxic effects of Cr, the combined treatment had the greatest effect. Mitigation of Cr intoxication occurred via reduced Cr uptake and translocation and by ameliorating reductions in water contents, light-harvesting pigments, and other photosynthetic parameters. In addition, the two hormones increased the activity of enzymatic and non-enzymatic defense mechanisms increasing the scavenging of reactive oxygen species, thereby reducing membrane damage and electrolyte leakage. Furthermore, the hormones reduced the accumulation of the toxic compound, methylglyoxal, by amplifying activities of glyoxalase I and glyoxalase II. Thus, applications of NO and EBL can significantly mitigate Cr-phytotoxicity when cultivating soybean plants in Cr-contaminated soils. However, further more-in depth studies including field investigations parallel with calculations of cost to profit ratios and yield losses are requested to validate the effectiveness of NO and/or EBL for remediation agents in Cr-contaminated soils with using key biomarkers (i.e., oxidative stress, antioxidant defense, and osmoprotectants) involved in the uptake, accumulation, and attenuation of Cr toxicity tested in our study.