Mass spectrometry (MS) provides a simple discrimination method for microorganisms. However, the presence of species such as melanin in fungal spores of Aspergillus niger (melanotic fungal) suppress ionization for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Inhibition of melanin synthesis pathways by tricyclazole enhances mycelium growth and protein contents of Aspergillus niger for four different media; namely sabouraud dextrose agar medium (SDA), potato dextrose agar (PDA), czapek dox agar (CDA) and yeast extract agar (YEA) media. The cell contents of protein and mycelium growth of Aspergillus niger are increased with the addition of a low concentration of tricyclazole (25–50 mg L−1). Furthermore, it improves the ionization signals of A. niger for MALDI-MS. This study reveals that inhibition of melanin using tricyclazole leads to the increase of protein content, mycelium …