Verticillium stripe, caused by Verticillium longisporum, is an emerging disease of canola (Brassica napus) in Canada. Studies were conducted to assess the impact of pH on both the growth of V. longisporum and its virulence on the canola host. Fungal growth was assessed by measuring the colony diameter following 14 and 21 days of incubation on potato dextrose agar at varying pH levels (4.7, 5.5, 6.5, 7.4, or 8.6). The results indicated that colonies of V. longisporum were approximately 16% greater in diameter at pH 7.4 and 8.6 compared with those at pH 5.5. The impact of pH on disease development at the seedling stage was investigated using a semi-hydroponic system with different pH levels of 4.4, 5.4, 6.3, 7.5, and 8.4 in half-strength Hoagland’s solution. Verticillium stripe was most severe at pH 7.5 and 8.4 after a 10-day period in the semi-hydroponic system. In a second inoculation experiment, canola seedlings previously inoculated with the fungus were transplanted into potting mix amended to four pH levels (5.6, 6.4, 7.2, and 7.8). The transplants were cultivated under greenhouse conditions and evaluated for Verticillium stripe severity at plant maturity. Disease severity was greatest at pH 7.8. This is the first study on the effects of pH on V. longisporum in canola. It suggests a substantial risk of increased disease severity and yield losses due to Verticillium stripe in regions with neutral to slightly alkaline soils.