Optimization of fermentation conditions is important for increased compound production and use of statistical designs could improve efficiency of the process. Optimization of nutritional and environmental requirements for increased GP-2B production by Geotrichum candidum OMON-1 in tryptose soy broth (TSB) at flask level using two-level factorial tool in Design Expert (DE) software was investigated. Using 1/8th factorial and design resolution IV, pre-determined low and high levels of tryptone, pH, peptone, sodium chloride, dextrose, agitation, fermenter volume and dipotassium hydrogen phosphate were considered in 16 randomized experimental runs. Antimicrobial activity (Response) of bioextract from each experimental run against Staphylococcus aureus was determined via agar well-diffusion. Fermentation proceeded in both DE-optimized and standard TSB medium and concentration (peak height) of HPLC-purified GP-2B compared. Highest antimicrobial activity (21 mm) was achieved at Run 13 (high tryptone (25 g/L), peptone (8 g/L), NaCl (25 g/L), agitation (150 rpm); low pH (5.5), dextrose (2 g/L), K2HPO4 (2 g/L), fermenter volume (40%)). Analysis of Variance (ANOVA) and Half-normal plot described tryptone (48.04%), tryptone-agitation interaction (19.85%), dextrose (-1.75; 12.01%) and pH (8.82%) as significant contributors to fermentation, while tryptone (+ 3.5) and dextrose (-1.75) had best and most detrimental influence respectively. A curved 3D response surface plot was obtained, with concentration and activity against S. aureus of GP-2B from optimized conditions (3.143 x 108; 21 mm) higher than at normal conditions (2.869 x 108; 19.5 mm). The two factorial design successfully improved fermentation requirements for increased GP-2B production by G. candidum OMON-1.