Background : Nasturtium officinale, belongs to the Brassicaceae, is a perennial plant growing in and around natural watersystem. It is commonly called watercress and commercially consumed as a salad crop in many countries. Hairy root cultures (HRCs), transformed by Agrobacterium rhizogenes (A. rhizogenes), have been noted for an experimental model system in plant metabolic engineering for the synthesis of natural products since hairy roots have fascinating properties including biochemical and genetic stability, high biosynthetic capacity for secondary metabolites and rapid growth rates. Methods and Results : The dry weights (DW) of watercress hairy roots was measured after 4-days freeze dryer. The highest weight was recorded after HRCs in SH and half-strength SH medium, followed by the levels of DW in MS and half-strength medium. However, the level of DW after HRCs in half-strength medium was lowest. SH medium is the most suitable for the growth of watercress hairy roots. Glucosinolate contents in the hairy roots varied responding to the basal media. 1/2 SH basal media resulted in the lowest value of total glucosinolate. MS basal media, however, resulted in the highest value of total glucosinolate, as well as the highest accumulation of each glucosinolate. The hairy roots cultured in 1/2 MS basal media showed the second highest value of total glucosinolate, followed by B5, SH and 1/2 B5. The accumulation of 4-Methoxyglucobrassicin and gluconasturtiin was also secondly higher in the hairy roots in 1/2 MS. In contrast, the level of 4-Hydroxyglucobrassicin and glucobrassicin were the second highest after culturing in SH and B5, respectively. Conclusion : In this study, media played a main role in growth and glucosinolate accumulation in watercress hairy roots. SH and half-strength SH media enabled the rate growth of hairy roots to be highest. In contrast, the highest accumulation of glucosinolate was recorded after HRCs in MS media. The current study suggests HRCs of watercress could be one of an effective alternative approaches for the enhanced production of glucosinolates