The use of perennial metal hyperaccumulators in phytoextraction provides an excellent gateway toward the removal of heavy metals from polluted sites, and the opportunity for the phytomining of valuable metals. In order to further advance our understanding of the effect of cropping systems on metal phytoextraction, it is important to investigate the effect of harvesting time. This study focuses on the variation in biomass production, Ni concentration and Ni mass across the different phenological stages, populations and organs of Alyssum lesbiacum, in order to evaluate when Ni phytoextraction is at a maximum. We sampled 60 single-phenological stage plots in three A. lesbiacum populations and we determined biomass production and Ni concentration at the plant organ level. Based on spontaneous A. lesbiacum vegetation, we were able to record remarkably high values of Ni phytoextraction. Biomass production and Ni concentration were found to be maximal on the third and fourth year of the A. lesbiacum life cycle respectively, while maximum phytoextraction capacity was reached in the third year. Our results: (1) demonstrate the significant variation in Ni phytoextraction across different phenological stages, populations and organs of A. lesbiacum, (2) imply that its phytoextraction potential is mostly influenced by biomass production and (3) suggest that perennial harvests could be an interesting alternative to consider in the future.