Publisher Summary This chapter provides an overview of nikkomycins and polyoxins. The detection, isolation, purification methods of nikkomycins and polyoxins are described in the chapter. The isolation procedure of polyoxins is similar to that of nikkomycins. The separation of polyoxins can be achieved with ion-exchange chromatography using strong cation exchange and strong anion exchange resins, followed by charcoal treatment and cellulose chromatography. Nikkomycins can be detected in biological cultures by the agar plate diffusion test, using the fungus Mucor hiemalis as test organism, which belongs to the zygomycetes and is especially sensitive against nikkomycins. Nikkomycins are hydrophilic compounds, and have one primary amino group and one or two carboxy groups. The whole spectrum of nikkomycins can be separated by ion-pair reversed-phase high-performance liquid chromatography (HPLC) using gradient elution. The antimicrobial activity of polyoxins can be determined against phytopathogenic fungi with agar plate diffusion assay using potato–sucrose medium. Polyoxins can be also detected by paper electrophoresis at pH 2 to 6.