Effects of high-pressure processing (HPP) pretreatment and inoculation with or without Lactobacillus senmaizukei (LS) on γ-aminobutyric acid (GABA) free amino acid content, glutamic acid (Glu) decarboxylase (GAD) activity, microflora, and physicochemical properties of the cocoa beans were investigated during fermentation, and microstructural changes of cocoa beans were observed through scanning electron microscopy (SEM). Raw cocoa beans had a GABA content of 21.12 mg/100 g, which increased to 31.26 mg/100 g after spontaneous (SPO) fermentation, while in HPP-LS fermented cocoa beans can be significantly increased to 66.35 mg/100g, due to higher GAD activity by ≥ 3-fold, which can improved the conversion efficiency of Glu to GABA. Therefore, the Glu content of HPP-LS cocoa beans dropped from 72.6 mg/100g to 26.1mg/100g at the end of the fermentation. Microbiological analysis showed that HPP-treatment altered the natural microbiomes of cocoa beans and reduced the counts of yeast and lactic acid bacteria. During the first 24 h of fermentation, yeast remained the dominant species in the presence/absence of LS inoculation. After 48 h, Lactobacillus spp. replaced yeast as the dominant species and acetic acid bacteria reached their maximum count after 120 h. After 72 h, the fermentation index of the HPP–LS cocoa beans was 1.02, whereas that of the SPO fermentation group reached 1.02 after 96 h; thus, HPP pre-treatment and LS inoculation reduced the fermentation time. HPP was shown to disrupt the microstructure of cocoa beans in SEM, increasing the probability of contact between GAD and Glu and upregulating GABA production. This findings can be used to evaluate the feasibility of producing GABA-enriched cocoa beans for functionality chocolate products development.