Climate change has challenged large-scale crop production at a global level. Global temperature increases, water scarcity, and a further reduction in cultivable land resources due to anthropogenic impacts have resulted in the need to redesign agricultural systems such as intercropping to maximize the efficient use of natural resources. Arbuscular mycorrhizal fungi (AMF) represent an underexplored area, not only in terms of an alternative to the heavy use of chemical fertilizers, but also as a natural resource used to enhance physiological processes and mitigate the variations in biotic and abiotic factors in plants. On the other hand, the combined use of AMF with suitable but cheaper and environmentally friendly growth substrates is another way to maximize crop production. A study was carried out in a tomato and onion intercropped pattern system to analyze the above- and belowground implications of two AMF commercial products containing Rhizophagus irregularis, propagated in soil and with an in vitro technique addition, with two different mixed growth substrates (river sand and compost) under greenhouse conditions. Overall, both AMF products overall showed significant promoting effects on plant growth (15–30%) and root parameters (50%) in the tomato and onion plants on the sand-mixed substrate. Moreover, the soil-propagated AMF also showed significant positive effects on chlorophyll content (35%), photosynthetic activity, and the accumulation of macro- and micronutrients, especially the Fe and Mn contents (60–80%) in the tomato plants. We present evidence of the benefits to plant performance due to the interactive effects between AMF and the growth substrate, and these positive effects might be due to the intercropping system. Hence, soil-propagated Rhizophagus irregularis is represented here as a promising candidate for enhancing growth, sustainability, and productivity under greenhouse conditions.