The use of inoculants is a critical strategy in legume production. In Zambia, inoculants are particularly used for the production of non-promiscuous genotypes of soya beans, but rarely for cowpeas and groundnuts. This study evaluated the response of soya beans, cowpeas and groundnuts to Biofix legume inoculants. Seeds were inoculated at the recommended or double the recommended rate at planting. Plants were grown under greenhouse conditions in a Completely Randomized Design for 7 weeks. Control, non-inoculated seeds were also planted and plants grown under the same conditions. At 7 weeks, nodule number and fresh weight per plant, nodule effectiveness (pinkness/redness), and above ground biomass were determined. Biologically fixed nitrogen was determined using the Nitrogen Difference Method. Nodule number and fresh weight per plant were higher at the recommended rate of Biofix application for soya beans and at both rates for groundnuts, while there was no effect on nodule fresh weight at either rate in cowpeas. All representative nodules assessed were effective. There was no significant benefit in inoculating seeds of the three legumes with Biofix on above ground biomass and biological nitrogen fixation. These results could suggest that possibly, the introduced strains though with a stronger nodulation competitiveness, were not as effective at fixing nitrogen as the indigenous strains in the soils in which soya beans, cowpeas and groundnuts have been repeatedly grown before. This could be an indication that sufficient and appropriate effective strains are already present in this soil. In general, the results suggest that to obtain the full benefits of biological nitrogen fixation, legume growers need to be provided with the correct inoculant, where required. Further work under field conditions is recommended to confirm these findings.