Iron (Fe) deficiency is one of the most common problems of soybean. It causes upper leaves yellowing, interveinal chlorosis, stunted growth and yield loss. Manganese (Mn) deficiency affects the reactions in the oxygen evolving complex (OEC) of photosystem II and increase the accumulation of reactive oxygen species (ROS). The aim of this research is to study the effect of Fe and Mn deficiencies applied separately and simultaneously on physiological, biochemical, nutritional and growth (morphological) parameters of soybean cultivars (Glycine max L.). The experiment was conducted in nutrient hydroponic solution lacking Fe or Mn or both Fe and Mn. Chlorophyll content index (CCI) and chlorophyll a fluorescence were measured through time to detect nutritional disorders at an early growth stage before the apparition of visual symptoms. The results showed that Fe and Mn deficiencies had a significant negative effect on the photosynthetic efficiency, CCI, stomatal conductance, protein content and shoot/root nutrient uptakes. Iron and manganese stress conditions were found to enhance the accumulation of secondary metabolites and increase the antioxidant activity such as total polyphenol content (TPC), malondialdehyde (MDA) and superoxide dismutase (SOD). These impacts were more accentuated when Fe and Mn stress were applied simultaneously than when any of the deficiencies was applied alone. More than that, Mn stress alone did not significantly affect the biomass accumulation. The obtained results showed that, in hydroponic conditions, iron and manganese rational fertilization can improve the studied parameters. •A significant negative effect on the photosynthetic efficiency, CCI, stomatal conductance, protein content and shoot/root nutrient uptakes.•Enhancement of the accumulation of secondary metabolites and increase the antioxidant activity such as TPC, MDA and SOD.•These impacts were more accentuated when Fe and Mn stress were applied simultaneously than when any of the deficiencies was applied alone. More than that, Mn stress alone did not significantly affect the biomass accumulation.•In hydroponic conditions, iron and manganese rational fertilization can improve the studied parameters.