Liamocins are unique extracellular glycolipid biosurfactants produced by certain species of with potential applications in agriculture, food, medicine, and pharmaceutical industries due to their surface-active properties and antimicrobial activity. In this study, for the first time, optimization of various fermentation parameters for liamocin production by NBRC 100716 strain was carried out using the response surface methodology (RSM). By conducting numeric optimization, the optimum conditions were determined as follows: initial substrate (fructose) concentration (X1) of 93.47 g/L, initial pH (X2) of 4.92, and temperature (X3) of 27.2 °C. Under optimum conditions, the maximum liamocin concentration (Y1), microorganism-specific growth rate (Y2), and maximum specific liamocin production rate (Y3) were found to be 3.56 g/L, 0.0670 h(-1), and 0.00450 [g liamocin/(g mo·h)], respectively. Variations of the responses with the independent variables were defined by a quadratic model. According to the analysis of variance (ANOVA) results, the model terms in the study showed high significance (p < 0.05) for all response variables, while the lack of fit was not significant for any of the responses. The coefficient of determination for liamocin production (0.829) was close to 1.0, confirming the significance of the model. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) determined that the liamocin structure contained all liamocin types referred to as A1, B1, A2, and B2. By the gas chromatography-flame ionization detection (GC-FID) method, it was determined that 61.12% of the fatty acid composition consisted of saturated and 38.87% consisted of unsaturated fatty acids (UFA). Furthermore, Fourier transform infrared spectrometry (FT-IR) spectral attributes were used for the first time to confirm the structure of liamocin, providing valuable insights. This study, in which some fermentation parameters were optimized, showed that the NBRC 100716 strain is promising for industrial-scale liamocin production in the future.