Biodegradation of a cotton-reactive dye (C. I. Reactive Blue 4, 50 mg/L) based on anthraquinone chromophore was attempted by a newly isolated bacterial species, Staphylococcus hominis subsp. hominis DSM 20328. The maximum dye decolorization efficiency was found to be 97% in 24 h upon optimization of pH, temperature, concentrations of carbon source and dye, salt, and glucose. Fourier Transform Infrared Spectroscopy analysis and High Pressure Liquid Chromatography was used to observe changes in the dye structure caused by the biodegradation, while the pathway for degradation was suggested by Gas Chromatography-Mass Spectrometry analysis. One of the metabolites formed was revealed to be catechol. Reduction in toxicity of the dye by formation of metabolites was established with the phytotoxicity and microtoxicity studies. Thus, the isolated bacterial species has a potential application in remediation of dyes and the decolorized wastewater can be safely disposed off into agricultural fields. To our knowledge, it is for the first time that a non-pathogenic bacterial strain of Staphylococcus species is being reported with decolorizing ability for anthraquinone chromophore-based dye along with the detailed mechanism of dye degradation.