The use of chemical fertilizers has led to significant environmental pollution. An alternative to these fertilizers is the use of natural compounds, such as phytohormones, which promote germination and crop development. However, environmental factors can affect natural compounds, reducing their effectiveness. Therefore, increasing their stability without decreasing their activity to improve crop quality is essential. This study produced and characterized chitosan and sodium tripolyphosphate (TPP) nano-microparticles (NMP) loaded with Bacillus subtilis extract and evaluated their impact on tomato seed germination. We employed two experimental designs (Box–Behnken and Box–Hunter–Hunter) to determine the optimal production conditions and characterized the NMP using DLS, SEM, and FTIR. The optimal treatment consisted of 8 min of homogenization, followed by 8 min of ultrasound at a 70% amplitude, resulting in a particle size of 330.7 nm, a polydispersity index of 0.25, a zeta potential of 34.3 mV, and an encapsulation efficiency of 68.8%. The NMP loaded with bacterial extract was applied to tomato seeds as a 50% dilution pretreatment. NMP achieved the best results, with a 72% germination rate (1.6 seeds per day) and an average germination time of 3.8 days. It is concluded that the experimental designs helped improve particle properties and that the chitosan and TPP coating enhances the stability and activity of the bacterial extract, potentially benefiting agronomic applications.