N and S play a significant role in plant growth and development and stimulate the production of secondary metabolites with anti-microbial properties. Under elevated CO2, plants raise their rate of carbon fixation causing increments across the synthesis of carbon that further lead to increased synthesis of carbon-based defence compounds and decrements across the concentrations of N and S in plant tissues. A pot experiment was performed in open-top chambers (OTCs) with ambient CO2 (390 ppm) and elevated CO2 (550 ppm), and mustard plants were supplemented with single doses of N and S separately as well as in combination. Furthermore, to monitor changes in defence ability, mustard plants were challenged with Alternaria brassicae and disease severity was assessed. Significant interactions were evident between CO2 and N and CO2 and S during the vegetative stage but not on the reproductive stage. Additive effects of both the nutrients and CO2 concentration were observed on physiological (net photosynthetic rate, stomatal conductance, sugars, proteins, C:N ratio) as well as defence markers phenylalanine ammonia-lyase enzyme and phenol concentration and disease severity. Combined nutrient treatment with N and S under elevated CO2 led to maximum enhancement of overall plant growth and defence in comparison to individual nutrient treatments. Mustard plants fertilized with N and S alone or in combination under enhanced CO2 improved plant's growth and defence.