Glabridin is a well-known active isoflavone found in the root of licorice (Glycyrrhiza glabra L.) that possess a wide range of biological activity. Plant cells, hairy roots, and fungal endophytes cultures are the most important alternative methods for plant resources conservation and sustainable production of natural compounds, which has received much attention in recent decades. In the present study, an efficient culture condition was optimized for the biomass accumulation and glabridin production from fungal endophyte Aspergillus eucalypticola SBU-11AE isolated from licorice root. Type of culture medium, range of pH, and licorice root extract (as an elicitor) were tested. The results showed that the highest and lowest biomass production was observed on PCB medium (6.43 ± 0.32 g/l) and peptone malt 5.85 + 0.11 g/l), respectively. The medium culture PCB was produced the highest level of glabridin (7.26 ± 0.44 mg/l), while the lowest level (4.47 ± 0.02 mg/l) was obtained from the medium peptone malt. The highest biomass (8.51 ± 0.43 g/l) and glabridin (8.30 ± 0.51 mg/l) production were observed from the PCB medium adjusted with pH = 6, while the lowest value of both traits was obtained from the same medium with pH = 7. The highest production of total glabridin (10.85 ± 0.84 mg/l) was also obtained from the culture medium treated with 100 mg/l of the plant root extract. This information can be interestingly used for the commercialization of glabridin production for further industrial applications.