The pH and substrate-to-inoculum ratio (S/I) are important parameters in the anaerobic fermentation of agroindustrial residues, and therefore the optimization of these two parameters is needed for a stable, efficient, and sustainable reactor operation. In this work, the parameters pH (5–9) and S/I (0.5–3 gVS gVS⁻¹) were optimized to produce biobased volatile fatty acids (VFAs) from hydrothermally pretreated olive mill solid waste (HPOMSW). The response variables evaluated in the Doehlert design were total VFAs concentration (tVFAs) (mg L⁻¹) and amounts (%) of isobutyric, butyric, isovaleric, and valeric acids on the VFAs profile. The pH was the variable that most influenced the mixed culture fermentation of HPOMSW, proving to be a key parameter in the process. Microbial community analyses of conditions 1 (S/I = 3 gVS gVS⁻¹ and pH = 7) and 4 (S/I = 1.13 gVS gVS⁻¹ and pH = 5) showed that Proteobacteria and Firmicutes accounted for more than 87% of the total microorganisms identified for both conditions. In addition, the second-order model best fitted the experimental data for the VFAs production at the desirable condition (S/I = 3 gVS gVS⁻¹ and pH = 8).