Gannan navel orange is a famous brand in China but the isolation of its endophytic fungi was rarely reported. In this study, a total of 54 strains of endophytic fungi were successfully isolated from the pulp, peel, twig, and leaf of Gannan navel orange; they were successfully identified to belong to 17 species of 12 genera. All these strains were fermented using potato-dextrose agar (PDA) medium, and their secondary metabolites were then extracted with ethyl acetate (EtOAc). The antibacterial assays ofEscherichia coli(E. coli), methicillin-resistantStaphylococcus aureus(MRSA), andXanthomonas citrisubsp.citri(Xcc) were also performed for the EtOAc extracts of these strains. As a result, the extracts of bothGeotrichumsp. (gc-1-127-30) andDiaporthe biconispora(gc-1-128-79) demonstrated significant antibacterial activities againstXcc, and the MIC value for the extract ofColletotrichum gloeosporioidesagainst MRSA was low to 62.5 μg/mL. Moreover, the chemical components of the extracts ofColletotrichumsp.,Diaporthe biconispora, andAnnulohypoxylon atroroseumwere primarily investigated, and they successfully led to the isolation of 24 compounds involving a new botryane sesquiterpene. Among the isolated products, compound2showed significant inhibitory activities toward SA, MRSA,E. coli, andXccwith MIC values of 12.5, 3.1, 125, and 12.5 μg/mL, respectively. This study revealed that the endophytic fungi of Gannan navel orange showed high potency to produce secondary metabolites with significant antibacterial effects.