The synergistic and antagonistic effects of arbuscular mycorrhizal fungi (AMF) and Azospirillum and Rhizobium nitrogen-fixers on the photosynthetic activity of alfalfa (Medicago sativa L.) were studied by means of the polyphasic chlorophyll a (Chl a) fluorescence transient O-J-I-P. The effects were evaluated on alfalfa plants grown in: (a) loamy chernozem control soil including the original rhizosphere community (bacteria+AMF); (b) gamma-sterilised soil (no microbes); (c) bacterial re-inoculated AMF-free soil (only bacteria). In these substrates the plants were inoculated with AMF (Glomus fasciculatum M107), with and without co-inoculation with the associative (Azospirillum brasilense Km5) and/or the symbiotic (Rhizobium meliloti Lu41+S5/7+K4/1) nitrogen-fixing bacteria. The Chl a fluorescence transients were recorded in vivo and analysed according to the JIP-test leading to the calculation of a constellation of parameters quantifying the photosystem II (PSII) behaviour. The beneficial effect of AMF is clearly revealed by the observed enhancement of the electron transport activity per leaf area. Based on the same criterion, an antagonism by both bacteria was detected. The antagonistic effect of Azospirillum was more pronounced than that of Rhizobium, though not strong enough to fully counter-balance for the beneficial effect of AMF. However, in the case of co-inoculation with both diazotrophs and AMF the electron transport activity was found to be only slightly lower than in the case of single inoculation by AMF, indicating that, in the presence of each other, the diazotrophs are no longer antagonistic to AMF. It was further clarified that these antagonistic effects are the net result of different synergistic and antagonistic effects, as follows: (a) Concerning the electron transport activity per absorption, Rhizobium has a synergistic effect on AMF's beneficial role, which is even more pronounced in the case of the tripartite co-inoculation, though Azospirillum in the absence of Rhizobium is antagonistic to AMF. (b) Concerning absorption per leaf, the beneficial influence of AMF is almost fully counterbalanced by co-inoculation with each of the diazotrophs or by both of them. The results demonstrate that the different combinations of inoculations and/or soils can be well distinguished by means of the JIP-test parameters and they thus suggest that the test can be used to screen, through the PSII behaviour of the plants, the effect of the microbial activity in the field. Two-dimensional rankings of the several soil/microbe combinations, in respect to the values of different energy fluxes, are shown to provide mappings that can be useful for the comparison of whole ecosystems or of individuals within an ecosystem.