High pressure/high temperature (HP/HT) and pulsed electric field (PEF) treatment of food are among the novel processing techniques considered as alternatives to conventional thermal food processing. Introduction of new processing techniques with fast and gentle processing steps may reveal new possibilities for preservation of healthy bioactive compounds in processed food. However, effects on various food components due to autolysis and fast reactions prior to the applied HP/HT or PEF need to be considered as the total contribution of processing steps affects the obtained food quality. The present experiments were performed on broccoli (Brassica oleracea var. Italica) florets, puree and juice. Specific focus was given to effects of HP/HT and PEF processing on the content of glucosinolates and activities of myrosinase isoenzymes (EC.3.2.1.147) in the broccoli preparations. Certain conditions applied in HP/HT processing of broccoli florets were able to maintain a high level of intact glucosinolates. Treatment at 700 MPa and 20°C for 10 min was found to inactivate myrosinase activity, but also pressure treatments at 300 MPa and 20°C were able to maintain a high level of intact glucosinolates present in the untreated broccoli florets. PEF processing of broccoli puree and juice showed that the myrosinase activities resulted in nearly total glucosinolate transformations as result of autolysis during pureeing and juice making prior to the PEF processing. These data demonstrated that insight into potential effects on myrosinase activities from application of PEF processing implies specific focus on the sample steps preceding the PEF processing. © by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences.