Particle size distribution in pre-treated organic substrates significantly impacts microbial fermentation efficiency, with smaller particles offering greater specific surface area, potentially reducing retention times and operational costs, particularly beneficial for remote and urban areas. Laboratory tests compared manual chopping, shredding, grinding, and mincing techniques, finding manual methods more effective than shredders at pretreatment level 1. Non-treated tomato waste showed the highest fine particle distribution (41%), contrasting with grass waste (< 2.9 mm), banana peel waste (< 2 mm), and paper waste (< 3 mm). Combining mincing, grinding, and extended processing increased methane output, particularly evident in higher pre-treatment levels with enhanced surface area. Among substrates, banana peel waste biomass (BPWSB) yielded the most methane (332 ± 36 Nml/gVS, 67% VSR), while grass waste biomass (GWSB) in semi-continuous tests produced 253 ± 29 Nml/gVS. The Biochemical Methane Potential (BMP) kinetic models consistently favored first-order kinetics over the modified Gompertz model across all substrates, attributing higher k-values to PTL4 due to its large surface area: BPWSB (0.59), GWSB (0.339), PWSB (0.59), and TWSB (0.59). These findings emphasized slower decay of larger particles and more rapid degradation of smaller fractions, crucial for optimizing biogas production efficiencies.