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Co-applying biochar and paper mill biosolids (PB) improves the potential of these by-products as soil amendments. Assessing the response of soil microbial communities to the co-application of biochar and PB is of great importance to further understanding their effectiveness. This hypothesis was tested in a 112-day incubation study performed separately on two acidic soils (Kamouraska clay and St-Antoine sandy loam) using two PB types (PB1 with C/ N = 24 and PB2 with C/ N = 13), co-applied with one of three rates (0%, 2.5%, and 5% w/w) of pine (Pinus strobus L.) biochar. An unamended control and a mineral-fertilized treatment were used as a reference. In the Kamouraska clay soil, co-applying 5% biochar and any PB increased microbial activity compared to PB-only application, as indicated by the fluorescein diacetate (FDA) hydrolysis assay. In both soils, co-applying biochar and PB did not affect microbial diversity and community structure compared with PB only. The application of any PB, however, increased microbial activity in both soils, while only PB2 shifted microbial community structure. Within each amendment and in both soils, microbial activity was lower at day 112 than at days 28 and 56, while major temporal shifts were observed only for bacterial community structure. Indeed, the difference in bacterial community structure between amendments was more pronounced at day 112. This study suggests that co-applying biochar and PB can improve soil properties than application of PB alone by increasing soil microbial activity without side effects on microbial diversity and community structure, especially in acidic clay soils. • Co-applying biochar and paper mill biosolids (PB) increased soil microbial activity. • Co-applying biochar and PB did not affect soil microbial community structure. • The incubation time affected soil microbial activity and community structure. [ABSTRACT FROM AUTHOR]