The effects of temperature and pressure processing on myrosinase extracted from fresh broccoli and brown mustard seed was studied. Brown mustard seeds had higher myrosinase activity (2.75 un/mL) than fresh broccoli (0.58 un/mL). The extent of enzyme inactivation increased with pressure (200-800 MPa) and temperature (30-80°C) for both brown mustard seeds and fresh broccoli myrosinase. However, at combinations of lower pressures (200-400 MPa) and temperatures (30-80°C), there was less myrosinase inactivation. When processing at a pressure of 300 MPa with a temperature of 70°C for 10 minutes, there was 65% myrosinase activity for brown mustard while at 300 MPa and 60°C, activity retention in fresh broccoli was 30%. Whereas, the corresponding activity retentions when applying only heat (70°C for 10 minutes) was 35% for brown mustard myrosinase, while there was no measurable myrosinase activity for fresh broccoli (60°C, 10 minutes). Thus, application of moderate pressures (200-400 MPa) on brown mustard and fresh broccoli can potentially be used to retain myrosinase activity needed for subsequent glucosinolate hydrolysis.