Arbuscular mycorrhizal fungi (AMF) are an essential component of agroecosystems as they provide multiple benefits to crops. However, they are critically affected by physical and chemical soil characteristics. Understanding the changes in abundance and composition of AMF communities during the multiplying process is important to optimize the inoculum generation methods and improve agricultural production and sustainability. Four substrates with different soil physicochemical properties: Soil + Sand (So + Sa), Soil + Vermiculite (So + Ve), Sand + Vermiculite (Sa + Ve), Soil + Sand + Vermiculite (So + Sa + Ve) were assessed to multiply three consortia of AMF from Coffea arabica and Plukenetia volubilis rhizospheric soil from San Martín. Fifteen species of AMF (ten genera and seven families) were identified. The species composition of the AMF community varied among the substrates; however, there were no differences in species richness. Substrate type significantly affected AMF spore production and mycorrhizal colonization (P ≤ 0.05). The So + Sa + Ve substrate presented a higher number of spores and percentage of colonization than the rest of the substrates. Paraglomus peruvianum was the most abundant species in the substrates So + Sa and So + Ve, while Rhizoglomus dunense was in the substrates Sa + Ve and So + Sa + Se. The properties of the substrates (OM content, pH, CaCO3, and EC) significantly modified the structure of the AMF community, allowing the occurrence of particular species in certain substrates. The composition of the substrates and their physicochemical characteristics influenced the composition and structure of the AMF community. For this reason, it should be considered when analyzing and multiplying the community of AMF present in agroecosystems, for use and conservation purposes.