Abstract. 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 F 1 broccoli hybrids, a factorial experiment based on completely randomized design in three replications was implemented at research station of Agricultural Sciences and Natural Resources University, Sari, Mazandaran, Iran in 2016. The results showed that mannitol stress reduced dry weight and shoot length significantly for all varieties but with a different rate. Total phenolic, flavonoid, and anthocyanin contents, the activity of enzymatic antioxidants, and DPPH activity were significantly higher under 176 Mm mannitol application condition than control. In addition, results showed that mannitol stress increased the content of sulforaphane. Marathon genotype showed the highest content of sulforaphane among all varieties under both normal (6.139) and under stress (14.122) conditions. Marathon and Heraklion genotypes could be suggestively used for breeding program to increase content of sulforaphane coupled with other traits. Since the content of sulforaphane along with phenolic compounds and antioxidant activities were higher under both severe and moderate stress conditions, using moderate mannitol stress treatment can be implemented for increasing the content of these suitable compounds in broccoli.