As glyphosate-resistant (GR) crops are becoming common in agro-ecosystems, their effects on non-target soil organisms need to be monitored. We evaluated soil microbial biomass C (MBC), bacterial functional diversity and community structure, and dehydrogenase enzyme activity in a field experiment conducted at six sites on the Canadian prairies. Treatments consisted of a factorial arrangement of three GR wheat and GR canola crop frequencies and two tillage systems. GR crop frequencies were arranged in 4-yr wheat-canola-wheat-pea rotations, with GR wheat and GR canola in zero of four, two of four, or three of four possible GR crop frequencies. The two tillage systems were either low soil-disturbance direct-seeding (LDS) or conventional tillage (CT). MBC increased with increasing frequency of GR crops in two of 20 site-years in the rhizosphere, and had no effects in bulk soil. Depending on tillage, GR crop frequency also affected the functional diversity of rhizosphere soil bacteria in only two of 20 site-years, and had no effects in bulk soil. Shifts in the structures of bacterial communities related to GR crop frequency were detected, but they were few and inconsistent. In three of 22 cases (10 in rhizosphere+12 in bulk soil), the activity of dehydrogenase enzyme decreased with increasing frequency GR crops in both the rhizosphere and bulk soil. In five of 20 site-years, soil MBC in the rhizosphere was greater under CT than under LDS, regardless of GR crop frequency. In bulk soil, tillage affected soil MBC in five site-years, three of which had greater MBC under CT than LDS, and vice versa in the other two. Tillage affected the functional diversity of soil bacteria in the rhizosphere in three site-years, but the effects were not consistent. Similar inconsistent tillage-related patterns were observed in the community structures of bacteria. There were no tillage effects on bacterial diversity in bulk soil. Dehydrogenase enzyme activity was greater under LDS than under CT in three of four cases in which tillage had significant effects. Overall, GR crop frequency effects on soil microorganisms were minor and inconsistent over a wide range of growing conditions and crop management.