In this work, the effects of light quality and beneficial microbes (biofertilizer) supply on structural and ecophysiological traits of spinach were investigated. Plants were grown under four light quality regimens: white light (WL), red-blue (RB), red-green (RG), and red (R) light, with or without the addition of biofertilizer. RG and R plants without biofertilizer showed morphological traits typical of shaded plants as wide leaf lamina and high photosynthetic pigment content. These plants also exhibited a higher photosynthetic capacity compared to WL and RB plants. The improved photosynthesis in RG plants was due to both morphological and physiological adjustments allowing a better utilisation of light energy, whereas in R plants it has been attributed to a reduced photorespiration rate. Biofertilizer application under WL improved plant performance enhancing photosynthesis. The high carbon gain compensates the costs of symbiosis. Biofertilizer application under R light favouring too much the microbial root colonisation, removed the benefits of symbiosis. The interaction of light quality and biofertilization significantly affects the root-microbe relationship.