Abstract Symbiotic ectomycorrhizal fungi have been shown to enhance host plant photosynthesis because the fungal symbiont are claimed to increase belowground carbon (C) demand. We aimed to study the effect of eight common symbiotic ectomycorrhizal (ECM) fungi on Scots pine photosynthesis and carbon (C) allocation because very few studies have investigated this phenomenon under controlled axenic conditions. The CO2 assimilation, stomatal conductance, chlorophyll fluorescence parameters, biomass allocation, moisture content, mycorrhization rate, nitrogen (N) uptake and N content of Scots pine seedlings, with or without symbiotic fungi, were measured after a four month growth period under axenic laboratory conditions. We hypothesized that ECM fungi are capable of increasing plant CO2 assimilation compared to non-mycorrhizal control fungi by increasing belowground C demand. This hypothesis was not supported since mycorrhizal and non-mycorrhizal plants did not differ significantly in any of the photosynthesis-related parameters. In addition, the common assumption that ECM fungi impose a C cost on the host plant was not supported by our study because mycorrhizal plants had the same or larger biomass compared to non-mycorrhizal plants. However, the mycorrhizal seedlings had a lower N percentage and allocated relatively more N to roots than shoots compared to control sterile seedlings, and the mycorrhizal seedlings had lower water content. In general, Needle N and water content correlated positively with CO2 assimilation. Based on our results, the N distribution within plant and plants water economy are the main effects of mycorrhizal symbiosis that affect plant C allocation and photosynthesis. These factors need to be considered when mycorrhizal effect on plant net primary production (NPP) also under natural conditions is estimated.