Biotic and abiotic stress are one of the most important factors affecting agricultural production. To assess the effect of drought stress induced by mannitol at three levels (0, 88 and 176 mM) on biochemical and polyphenolic traits of six F₁ broccoli hybrids, (Green Magic, Sacura, Heraklion, Marathon, Matsuri and Castell Dom) a factorial experiment based on completely randomized design was implemented at research station of Agricultural Sciences and Natural Resources University, Sari, Mazandaran, Iran in 2016. The results showed that using mannitol as drought stress reduced dry weight (10-25%) and stem length (12 to 30%). The activity of antioxidant enzymes averaged 19 to 230 and the activity of 2,2-diphenyl-1-perryl hydrazil, ȳ=3 Was significantly  in 0.05 level increased in mannitol at 176 mM compared with control. Also, the results showed that mannitol stress increased the amount of sulforaphane, so that the response of sulforaphane to the stress levels was different in different genotypes. Marathon showed the highest sulphoraphane content in all genotypes under normal conditions (6.139) and under stress (14.122). The results of path analysis showed that phenol and some antioxidant enzymes had a direct and significant relationship with sulforaphane in both normal and stress conditions. The Principal Component Analysis showed that 73.5% in normal conditions and 68% in stress conditions from total variation were explained by the first two components. Biplot results categorized the genotypes into three groups. In terms of stress, sulphoraphane and antioxidant enzymes and marathon genotype in the first group, anthocyanin, flavonoids and malondialdehyde, and genotypes of Heraclion and Green magic in the second group and 2, 2-diphenyl-1-perryl hydrazyl, dry weight and stem length, and Genotypes of Matsuri and Castell Dom were placed in the third group. As a result, marathon was the most suitable genotype for economic cultivation in agricultural fields.