Biochar and organic fertilizer are widely supported to maintain crop production and sustainable development of agroecosystems. However, it is unclear how biochar and organic fertilizer alone or in combination regulate soil functional microbiomes and their relationships to ecosystem multifunctionality (EMF). Herein, a long-term (started in 2013) field experiment, containing five fertilization treatments, was employed to explore the effects of biochar and organic fertilizer applications on the EMF (based on 18 functional indicators of crop productivity, soil nutrient supply, element cycling, and microbial biomass) and the functional microbiomes of bulk soil and rhizosphere soil [normalizing the abundances of 64 genes related to carbon (C), nitrogen (N), phosphorus (P), and sulphur (S) cycles]. Compared with single-chemical fertilization, biochar and organic fertilizer inputs significantly enhanced most ecosystem-single functions and, in particular, the EMF significantly increased by 18.7–30.1%; biochar and organic fertilizer applications significantly increased the abundances of soil microbial functional taxa related to C-N-P-S cycles to varying degree. The combined application of biochar and organic fertilizer showed a better improvement in these indicators compared to using them individually. Most functional microbial populations in the soil, especially the taxa involved in C degradation, nitrification, nitrate-reduction, organic P mineralization, and S cycling showed significantly positive associations with the EMF at different threshold levels, which ultimately was regulated by soil pH and nutrient availability. These results highlight the strong links between soil microbiomes and agroecosystem functions, as well as providing scientific support for inclusion of biochar in agricultural production and services with organic amendments. Graphical Abstract