The significant neutralization of waste streams required after the acidic bio-oxidation of sulfidic gold ores could be avoided by performing a novel treatment at circumneutral pH with an in situ neutralization. For the first time, the white-rot fungus Phanerochaete chrysosporium was incubated in a modified culture medium containing corn steep, an industrial waste product, to support microbial activity and, subsequently, the oxidation of a sulfidic ore at an initial circumneutral pH environment. In this investigation, the concentration of the native culture medium ingredients was first evaluated with response surface methodology to attain maximum sulfide oxidation. The statistical analysis proposed a modified culture medium composed of 12.86 g/L glucose, 2.20 g/L malt extract, 1.67 g/L yeast extract, and 0.49 g/L MgSO4·7H2O to reach a maximum of 28.7% sulfide oxidation after 14 d-bio-oxidation. pH-controlled batch cultures showed that an increase in initial pH in the range of 5.8 to 7.0 reduced the microbial activity, affecting sulfide oxidation. In addition, the modified culture medium at which yeast extract was substituted with 1.67 g/L corn steep produced comparable microbial activity and sulfide oxidation after 14 d, attaining 21.6% at 5% w/v with a maximum 39 U/L lignin peroxidase and 116 U/L manganese peroxidase. A 40.6% sulfide oxidation and 43.8% gold recovery were obtained after 42 d three-cycle replenishing bio-oxidation and 24 h cyanidation, respectively. Overall, corn steep waste showed the potential to substitute more expensive culture medium ingredients, supporting microbial activity and oxidation of sulfidic gold ores at an initial circumneutral pH and contributing to circularity of waste management.