Inoculation with Azospirillum brasilense has promisingly increased plant yield and nutrient acquisition. The study aimed to estimate the dose of A. brasilense that increases yield, gas exchange, nutrition, and foliar nitrate reduction. The research was carried out in a greenhouse at Ilha Solteira, in a hydroponic system in randomized blocks with four replicates. The treatments consisted of doses of inoculation with A. brasilense strains AbV5 and AbV6 via nutrient solution (0, 8, 16, 32, and 64 mL 100 L−1). Inoculation with A. brasilense at calculated doses between 20 and 44 mL provided the highest fresh and dry mass of shoots and roots, number of leaves, and leaf yield. In addition, the calculated doses of inoculation with A. brasilense increased the accumulation of N, P, K, Ca, Mg, S, B, Fe, Mn, and Zn in shoots and roots, except the accumulation of Ca in roots. It also increased cell membrane integrity index (15%), relative water content (13%), net photosynthesis rate (85%), intracellular CO2 concentration (15%), total chlorophyll (46%), stomatal conductance (56%), transpiration (15%), and water use efficiency (59%). Hence, inoculation with A. brasilense at doses between 20 and 44 mL 100 L−1 is considered the best approach for increasing the growth, yield, accumulation of nutrients, and gas exchange of hydroponically grown iceberg lettuce.