The effects of biochar on soil–plant–microorganisms systems are currently being extensively investigated. Considering that arbuscular mycorrhizal fungi (AMF) play an essential role in nutrient dynamics, the present study aims at understanding vine shoot-derived biochar effects on AMF activity and the impact of their multiplication in soils on water-stress resistance of plants. Three agronomic tests were performed in greenhouse pots. The first experiment evaluated the effects of three factors: final pyrolysis temperature for biochar production (400 °C and 600 °C), application rate (0 weight-wt.- % as a control, 1.5 wt. %, and 3.0 wt. %) and texture of the growing media (sandy-loam and clay-loam origin) on AMF, microbial communities and phosphatase activity. In the second experiment, an indigenous consortium of AMF was multiplied through the solid substrate method and sorghum as a trap plant with biochar addition. This process was compared to a control treatment without biochar. Obtained inocula were tested in a third experiment with lettuce plants under different water irrigation conditions. Results from the first experiment showed a general increase in AMF activity with the addition of the biochar produced at 400 °C in the sandy-loam texture substrate. Results of the second experiment showed that the biochar addition increased AMF root colonization, the number of AMF spores and AMF infective potential. Results of the third experiment showed that biochar-derived AMF inoculum increased AMF root colonization, AMF spores, dry biomass and the SPAD index in a lettuce crop under low-water irrigation conditions.