Nickel (Ni) agromining of ultramafic soils has been proposed as an eco-friendly option for metal recovery, which can also improve the fertility and quality of these low productive soils. The selection of adequate plant species and the analysis of their performance under the different climatic conditions are of interest for optimising the process and evaluating its full viability. A one-year field experiment was carried out to evaluate the viability of the two Ni-hyperaccumulating Mediterranean species, Alyssum murale and Leptoplax emarginata, for agromining purposes in ultramafic soils under a humid-temperate climate. Field plots of 50 m2 were established and the soil was fertilised with gypsum and inorganic NPK fertilisers prior to cropping. Alyssum murale produced a slightly higher Ni yield than L. emarginata, but Ni bioaccumulation was dependent on the plant phenological stage for both species, being maximal at mid-flowering (4.2 and 3.0 kg Ni ha−1, respectively). In both species, Ni was mainly stored in the leaves, especially in leaves of vegetative stems, but also in flowers and fruits in the case of L. emarginata. The main contributors to Ni yield of A. murale were flowering stems and their leaves, while for L. emarginata they were flowering stems and fruits. Implementing the agromining system increased soil nutrient availability, and modified microbial community structure and metabolic activity (due to fertilisation and plant root activity). The soil bacterial communities were dominated by Proteobacteria, Actinobacteria, Acidobacteria and Chloroflexi, and the agromining crops modified the relative abundance of some phyla (increasing Proteobacteria, Bacteroidetes and Nitrospirae and reducing Acidobacteria and Planctomycetes). Cultivating A. murale increased the densities of total culturable bacteria, while L. emarginata selected Ni-tolerant bacteria in its rhizosphere. In summary, both species showed great potential for their use in Ni agromining systems, although optimising soil and crop management practices could improve the phytoextraction efficiency.