Endophytes are important components of forest ecosystems, and have potential use in the development of medical drugs and the conservation of wild medicinal plants. This study aimed to examine the diversity and antimicrobial activities of endophytic fungi from a medicinal plant, Litsea cubeba (Lour.) Pers. The results showed that a total of 970 isolates were obtained from root, stem, leaf, and fruit segments of L. cubeba. All the fungal endophytes belonged to the phylum Ascomycota and could be classified into three taxonomic classes, nine orders, twelve families, and seventeen genera. SF15 (Colletotrichum boninense) was the dominant species in L. cubeba. Leaves harbored a greater number of fungal endophytes but lower diversity, while roots harbored the maximum species diversity of endophytic fungi. For the antimicrobial activities, seventeen isolates could inhibit the growth of plant pathogenic fungi, while the extracts of six endophytes showed antimicrobial activity to all the tested pathogenic fungi. Among these endophytes, SF22 (Chaetomium globosum) and SF14 (Penicillium minioluteum) were particularly effective in inhibiting seven plant pathogenic fungi growths and could be further explored for their potential use in biotechnology, medicine, and agriculture.