Biochar and paper mill biosolids (PB) are reported to improve soil fertility, crop growth and indirectly conditions for soil microbial communities. However, it is unclear how the co-application of these materials impacts soil microbial communities under field conditions. A study was initiated in 2018 in Québec, QC, Canada and fine roots and rhizosphere soil were sampled to determine the effects of co-application of wood biochar (0, 10, and 20 Mg dry wt. ha−1) and PB (0 and 30 Mg wet wt. ha−1) on the percentage of corn (Zea mays L.) and soybean (Glycine max L. Merr.) root colonization by arbuscular mycorrhizal fungi (AMF), soil microbial biomass phosphorus (MBP) and microbial (Bacteria, Fungi and AMF) diversity and community structure in a temperate loamy soil. Co-applying PB and biochar increased soil MBP compared with the control and biochar-only application. Applying PB alone or with biochar increased the level of root colonization by AMF compared with the control and biochar-only application in soybean but not in corn. Overall, biochar and PB application had no significant effect on bacterial and AMF diversity and community structure compared with the control. However, applying PB alone or with biochar decreased the fungal alpha diversity (Shannon and Simpson indices), affected several fungal taxa abundances and shifted the fungal community structure as indicated by the principal coordination analysis (PCoA). Our results provided an understanding on the short-term effects of co-applied wood biochar and PB on microbial communities of a temperate loamy soil under field conditions, as well as scientific bases for further investigation.