This work studied the influence of culture medium composition and pH on exopolysaccharide (EPS) production by Pleurotus sajor-caju and validates the antitumor activity of the produced EPSs and of the mycelial biomass (intracellular polysaccharides [IPSs]) against Sarcoma 180 (S180) cells. The effect of the initial concentrations of (NH₄)₂SO₄, yeast extract and soy peptone on EPS production by P. sajor-caju was studied in shake flasks. A bioreactor was used to evaluate the pH values and the initial CaCO₃ and glucose concentrations. Extracts of EPSs (PE1) and IPSs obtained through two different separation processes (PM1 and PM2) were tested on mice inoculated with S180 cells. A medium containing 2.5, 1.0, and 1.0 g/L of (NH₄)₂SO₄, yeast extract and soy peptone, respectively, provided the highest EPS concentration (0.6 g/L). The use of pH 4.0, 1.0 g/L CaCO₃ and 20 g/L initial glucose concentration enhanced EPS productivity (3.84 g/L per hour). The PE1 extract promoted the highest reduction of S180 growth (86%), followed by the PM2 extract (80%); growth reduction was dose-independent for both substances. This work provides information about culture medium and conditions that enhanced the production of extracellular polysaccharides by P. sajor-caju. The results can contribute to the search for new bioactive products bringing novel aspects to the medical and pharmaceutical areas.