This study presents an in vitro evaluation of the antitumor potential of a chitin-like exopolysaccharide (EPS, produced by Mortierella alpina) on Adrenocortical carcinoma cells (ACC) compared to mitotane, a commercial drug commonly used in ACC treatment, and known for its side effects. Techniques of cellular viability determination such as MTT and fluorescence were used to measure the cytotoxic effects of the EPS and mitotane in tumoral cells (H295R) and non-tumoral cells (VERO), observing high cytotoxicity of mitotane and a 10% superior pro-apoptotic effect of the EPS compared to mitotane (p < 0.05). The cytotoxic effect of the EPS was similar to the effect of 50 μM mitotane on tumoral cells (p < 0.05). A decrement of the lysosomal volume was also noted in tumoral cells treated with the EPS. To enhance the antitumor effect, a combination of mitotane at a lower dosage and the EPS (as adjuvant) was also tested, showing a slight improvement of the cytotoxicity effect on tumoral cells. Therefore, the results indicate a cytotoxic effect of the EPS produced by Mortierella alpina on adrenocortical carcinoma, and a possible application in biomedical formulations or additional treatments.