The application of arbuscular mycorrhizal fungi (AMF) in a mixed substrate is a vegetable production practice capable of improving plant productivity and sustainability of agricultural ecosystems. This study was conducted to determine the effect of a mixed substrate (agricultural soil with coconut fiber substrate) not enriched (S0) and enriched with 100 and 300 g m−3 of the fungus Glomus iranicum var. Tenuihypharum (S100 and S300) in water relations, photosynthetic and nutritional capacity, as well as production and fruit quality in three tomato varieties “H4”, “H3” (susceptible to mycorrhization) and “Jacaranda” not susceptible to mycorrhization). Plants irrigated with moderately saline water (EC: 2.35 dS m-1) in a commercial greenhouse in Mediterranean climate conditions were used for this study. Results showed that plants grown in substrate S100 showed an increase of AMF colonization, leaf phosphorus concentration, leaf total carbon and total nitrogen content compared with those grown in S0 and S300, which induced an increase in photosynthesis and stomatal conductance. This positive response was less evident by the application of substrate S300. Variety “H3” had a decrease in leaf sodium content, as well as higher photosynthesis values. There were no differences in stem water potential between factors. The lowest fruit production was observed in the “H4” variety, while fruit firmness decreased by AMF concentration and in variety “Jacaranda”. Root and aerial biomass increased in plants grown in S100 and S300, with respect to S0. Therefore, the improvements in the physiological and nutritional behaviour of these plants resulted in increased vegetative growth, but not in increased production. Varieties susceptible to AMF infection were more colonized than “Jacaranda”, but there were not improvements in yield and fruit quality. These results suggest that aspects related to the composition of the selected substrate, enzymatic AMF activity, fruit nutrition, as well as molecular mechanisms that control the plant-AMF interaction should be studied in depth.