Conventional high-temperature calcination for activating MFI zeolite membranes is energy-intensive and prone to inducing defects. Here, we demonstrate that a rapid ozonation treatment at 200 °C for only 1 h effectively decomposes organic templates while preserving membrane integrity. The resulting membrane exhibits H₂/CH₄ and H₂/N₂ ideal selectivities of 10.3 and 6.5, respectively, at room temperature, with C₃H₈ and SF₆ permeances below the detection limit. These results confirm a dense, defect-minimized architecture and good molecular sieving performance of the zeolite membrane. In contrast, extending ozonation to 48 h leads to defect formation and a marked reduction in selectivity. For H₂/CH₄ mixture separation, the membrane achieves a selectivity of 23.8 at 100 °C, which is highly competitive among reported MFI membranes. In isopropanol dehydration, it achieves a water flux of 2.3 kg·m⁻²·h⁻¹ and a separation factor of 3278 at 70 °C with a 10 wt% water feed, while maintaining >99.5 wt% water content in the permeate over a broad operating temperature range (30–70 °C). This work establishes rapid ozonation as a scalable, energy-efficient activation method for high-performance MFI zeolite membranes in both gas and liquid separations.