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Nerium oleander is an important medicinal plant. In order to study the diversity of endophytic fungi from N. oleander and evaluate the activity of its secondary metabolites, the endophytic fungi of N. oleander in Guangxi were isolated and purified, and identified by a combination of morphology and ITS sequence analysis. Then the antibacterial activities of endophytic fungus extracts were screened with five indicator bacteria (including three Vibrio species). The results were as follows: (1) A total of 19 endophytic fungi were obtained from Guangxi N. oleander. The ITS sequence analysis showed that these 19 endophytic fungi all belonged to the Ascomycota, and covered five orders and seven genera, which including Colletotrichum, Guignardia, Phyllosticta, Neofusicoccum, Aspergillus, Nothophoma and Diaporthe. Among them, the dominant genera were Colletotrichum (the separation rate was 36.85%) and Guignardia (the isolation rate was 21.05%). Colletotrichum was mainly distributed on stems, and Guignardia all originated from leaves. (2) Antibacterial experiments showed that jing-117 (Neofusicoccum sp.) and ye-130 (Guignardia sp.) had specific antibacterial effects on Vibrio campbellii, ye-136 (Aspergillus sp.) could simultaneously inhibit Bacillus cereus and Vibrio campbellii. ye-135 (Aspergillus sp.) and jing-116 (Colletotrichum sp.) could only inhibit B. cereus, and ye-134 (Guignardia sp.) had an inhibitory activity to Vibrio alginolyticus. The study revealed for the first time that endophytic fungi of N. oleander in Guangxi has a relatively rich diversity based on the ITS sequence. Some antibacterial active strains could be screened, and their extracts could inhibit the growth of aquatic pathogen Vibrio, which will has good development value in the future.