Fungal endophytes have been found to exist in many plant species and appear to be important to their plant hosts. However, the diversity and biological activities of these fungi remain largely unknown. Zanthoxylum simulans Hance, a popular natural spice and medicinal plant, commonly known as Szechuan pepper or Chinese-pepper, grows on Kinmen Island, Taiwan. In this study, leaf and stem samples of Z. simulans, collected in summer and winter, were screened for antimicrobial and anti-inflammatory metabolite-producing endophytic fungi. A total of 113 endophytic strains were isolated and cultured from Z. simulans, among which 23 were found to possess antimicrobial activity, belonging to six fungal genera: Penicillium (26.09%, 6), Colletotrichum (21.74%, 5), Diaporthe (21.74%, 5), Daldinia (17.39%, 4), Alternaria (8.70%, 2), and Didymella (4.34%, 1). We also found that the number of species with antimicrobial activity and their compositions differed between summer and winter. Our study demonstrated that Z. simulans might contain large and diverse communities of endophytic fungi, and its community composition varies seasonally. In addition, fungal endophytes produce antimicrobial agents, which may protect their hosts against pathogens and could be a potential source of natural antibiotics.