A melanin-producing strain was identified as Streptomyces sp. ZL-24 by phylogenetic analysis of 16S rRNA gene sequences. Based on single factor experiment, NiCl, FeSO and soy peptone were selected as significant variables of melanin production. Subsequent biochemical and RT-qPCR analyses revealed that melanin production was markedly driven by moderate levels of ferrous and nickel ions. The central composite design indicated that the optimal medium was composed of 3.05 mM NiCl, 1.33 g/L FeSO and 20.31 g/L soy peptone. The maximum production of the insoluble melanin (189.90 mg/L) was obtained using 3% (v/v) inoculation size in the medium of pH 7.0 at 30 °C for 5 days. Under this optimal condition, an indigenous plate culture system was standardized and used to produce soluble melanin. The resulting pure melanin pigment was up to 4.24 g/L, which set a new record of soluble melanin production. Bioactivity assay in vitro showed that both melanin pigments had strong antimicrobial, anti-biofilm and antioxidant activity. To our best knowledge, the results provide novel data on soluble melanin production, and identify an excellent candidate for anti-biofilm application.