The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro, but the details of these interactions are poorly understood and their implications on in vivo antifungal activity have not been established. Here, we explore how the coordination environment of Cu influences chemical and biological activity of the antifungal peptide Hist-5. Antifungal susceptibility assays and Cu-binding experiments reveal how the efficacy of Hist-5 against the commensal organism Candida albicans depends on the availability of Cu in the growth environment. Further, this biological activity correlates with the presence of adjacent histidine residues (bis-His) within the histatin peptide that support Cu(I) binding in the low nM range. Evaluation of oxygen reactivity of the Histatin Cu(I)-bis-His complexes indicates the PCu(I) complex is reactive towards H2O2. EPR, UV-Vis and HPLC studies demonstrate that exposure to H2O2 results in the formation of a metalloradical complex reminiscent of radical copper oxidases. Additional exploration of the coordination environment conducive to metalloradical formation exposes the importance of the third ligand (His3) of the Cu(I)-bis-His Complex for H2O2. His3 mutant peptides also disclose the tunability of the H2O2 reactivity. Furthermore, substrate evaluation assays offer evidence of the capability of the Cu-Hist-5 to specifically chemically modify a cell wall component. Together, these results provided compelling evidence supporting that Cu-coordination plays a critical role in the biological and chemical activity of Hist-5.