In this study, it was shown that the amount of available water was found to influence the high pressure processing (HPP) effect on both myrosinase activity and total glucosinolate concentration in Brussels sprouts seedlings. Brussels sprouts seedlings with different water content (w_c = 4.8–89.4%) and water activity (a_w = 0.17–0.97) were pressurized at selected pressures between 200 and 800 MPa (5 °C and 3 min), thereby affecting pressure-induced enzyme denaturation, molecular diffusion, and cell permeability differently. The myrosinase activity and intact glucosinolate content in the dry seedlings (w_c < 14%) was not affected by the applied pressure treatments. Water adsorption (duration of 5 days) prior to HPP resulted in a decreased initial myrosinase activity due to changed plant cell permeability. Myrosinase was inactivated in seedlings with high water availability (w_c = 45–89%) after HPP, this inactivation is interpreted to be both pressure-induced and result from glucosinolate product catalyzed inactivation facilitated by enhanced cell permeability. Industrial relevance: High pressure processing (HPP) is increasingly applied in the food industry. The treatment is acknowledged for the ability to give products longer shelf life concomitant with a high nutritional quality and fresh appearance. Upon HPP of cruciferous plants it is important to have a special focus on the glucosinolate-myrosinase system, since sub-optimal pre-treatment and/or processing parameters can affect the food quality negatively. The present study provides valuable results regarding the significance of water content and activity on the sensitivity of the myrosinase-glucosinolate system in Brussels sprouts seedlings towards HPP. Thus, providing a tool for designing different types of HPP products with respect to levels of active myrosinase and intact glucosinolates by adjustment of water content, water activity and HPP level.