Mesquite seed (Prosopis africana) is an underutilized tropical seed with nutritional and economic potential and is traditionally processed into fermented condiments. Because traditional fermentation is carried out under different local conditions, the progress and outcome of the process can vary. This study examined fermentation time, temperature and particle size as controllable factors and used response surface methodology (RSM) to relate them to nitrogen evolution. Matured mesquite seeds obtained from Afor-Opi, Nsukka Local Government Area, Enugu State, Nigeria, were boiled, decarped, oven-dried at 35°C, grated and sieved into particle sizes of 250–1200 µm. Fifty-gram portions were fermented at 25–45°C for up to 120 h, and nitrogen evolution was monitored at 24-h intervals. The RSM design produced 20 randomized experimental combinations for the mesquite seed. The resulting quadratic model was significant (F = 153.12, p < 0.0001) and accounted for 99.28% of the variation in nitrogen evolution (R² = 0.9928). The adjusted R² and predicted R² were 0.9863 and 0.9456, respectively, while adequate precision was 52.011. Fermentation time, temperature, particle size, the time–temperature interaction, the time–particle-size interaction and the quadratic time term were significant. Numerical optimization gave 113.6 h, 44.33°C and 313.33 µm as the best combination within the experimental region, with a predicted nitrogen evolution of 0.915%. The result indicates that the three operating variables can be used together to control the fermentation response. The predicted condition should, however, be confirmed experimentally before it is considered for scale-up.