C. brachyspora, a widespread dematiaceous fungus, was evaluated in this study to optimize the production of exopolysaccharides (CB-EPS). Optimization was performed using response surface methodology, and the best production yielded 75.05% of total sugar at pH 7.4, with 0.1% urea, after 197 h. The obtained CB-EPS showed typical signals of polysaccharides, which was confirmed by FT-IR and NMR. The HPSEC analysis indicated a polydisperse polymer, showing a non-uniform peak, with an average molar mass (Mw) of 24,470 g/mol. The major monosaccharide was glucose (63.9 Mol%), followed by mannose (19.7 Mol%), and galactose (16.4 Mol%). Methylation analysis encountered derivatives that indicated the presence of a β-d-glucan and a highly branched glucogalactomannan. CB-EPS was tested on murine macrophages to verify its immunoactivity, and the treated cells were able to produce TNF-α, IL-6, and IL-10. However, the cells did not produce superoxide anions or nitric oxide nor stimulated phagocytosis. The results demonstrated an indirect antimicrobial activity of macrophages by stimulating cytokines, showing another biotech applicability for the exopolysaccharides produced byC. brachyspora.