Colletotrichum gloeosporioides (C. gloeosporioides) is widely distributed pathogen responsible for postharvest losses in fruits and vegetables. Black pepper essential oil (BPEO) has emerged as natural antifungal agent that effectively inhibits C. gloeosporioides growth. However, the precise mechanism remains incompletely understood. This research investigates the suppressive properties of BPEO nanoemulsion on C. gloeosporioides and its impact on the respiratory metabolism. Results indicated that 2 MIC BPEO nanoemulsion have the best antifungal effect. Mechanistically, 2 MIC BPEO nanoemulsion can better suppress key enzymes connected to the Tricarboxylic Acid (TCA) cycle and Embden-Meyerhof-Parnas (EMP) pathway, hence reducing the C. gloeosporioides respiration rate. The total respiratory rate decreased by 60.54%, after 2 MIC BPEO nanoemulsion treatment. In conclusion, the BPEO nanoemulsion effectively mitigates fungal proliferation and reproduction by downregulating relevant enzymes expression. However, it is necessary to further expand the research on the regulation mechanism of enzyme activity at gene and protein level.