Abstract Modulation of the water availability for the plants can be a sustainable option to reduce agricultural water demand and stimulate the synthesis and accumulation of secondary metabolites. However, information on the reduction of water availability while maintaining yield and functional quality are scarce and fragmentary. This research aimed at elucidating the agronomical, physiological and functional quality (ascorbic acid, phenolic acids and flavonoids) responses triggered by a microbial-based biostimulant containing two strains of arbuscular mychorrizal fungi (AMF) and Trichoderma koningii of greenhouse grown lettuce (Lactuca sativa L.) under well watered (WW), moderate (MDI) or severe deficit irrigation (SDI) regimes. Reducing water availability from WW to MDI did not affect yield, phenolic acids and flavonoids concentration, but reduced both AMF and Trichoderma presence in the roots and soil, respectively, along with plant Mg and Zn concentration by 12.4% and 26.8%, respectively, and almost halved net photosynthetic rate and transpiration. Further reduction in water availability also reduced yields, along with ascorbic acid, total phenols and quercetin. The biostimulant application increased a wealth of traits, including P, Mg, Fe, Mn, and Zn by 20.8%–97.4% and various phenolic acids compared to the non-inoculated control. This effect occurred irrespective of the water availability. In addition, the microbial-based biostimulant increased plant yield, Ca and Cu, and isochlorogenic acid concentrations, but such effects were evident under WW and MDI, only. Luteolin glycoside, that is frequently associated to a plant reaction to water deficit but also to a microbial stimulation, did not vary at reducing water availability in non-inoculated control and progressively increased in the biostimulant inoculated plant. These results suggest that the biostimulant effect on lettuce nutritional and functional quality was mostly independent of the water availability, whereas its effect on fresh marketable and dry yields were evident in WW and MDI, only, through a modulation of the biosynthesis of secondary compounds rather than nutrient uptake. These results have a high practical implication when aiming to improve both plant yield and product quality while reducing water availability.