Abstract Lasiodiplodan, an exopolysaccharide of the (1→6)-β-d-glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO3, (NH4)2SO4, urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan’s anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 ± 0.05 g L−1), maltose (2.08 ± 0.04 g L−1) and yeast extract (2.46 ± 0.06 g L−1) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 ± 0.006 vs. 0.22 ± 0.008 g g−1). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 ± 0.07 g L−1) with a specific yield of 0.25 ± 0.57 g g−1 and a volumetric productivity of 0.06 ± 0.001 g L−1 h−1. A factorial 22 statistical design developed to evaluate the effect of glucose concentration (20–60 g L−1) and impeller speed (100–200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L−1) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60°C in the presence of CaCl2. Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC50 of 100 μg lasiodiplodan mL−1.