Aim This study investigated the biodiversity of fungi associated with the pistachio bark beetle, Chaetoptelius vestitus, in Tunisia. We evaluated the phytopathogenic activities and tested antagonistic potentialities with respect to phytopathogens. Methods and Results A total of 41 fungal isolates were randomly isolated from C. vestitus adults and galleries. We identified 28 species belonging to 13 genera using ITS sequences of the ribosomal RNA operons. Pathogenicity assays performed using the excised shoot method revealed that isolates Aal_io_1 (Alternaria alternata), Feq_io_1 (Fusarium equiseti), Fgra_io_1 (Fusarium graminearum), Fve_io_1 (Fusarium verticilloides), Tro_io_1 (Trichothecium roseum) and Nqu_io_1 (Nothophoma quercina) displayed a high pathogenic activity on pistachio stems. Estimation of the antagonistic potentialities of isolated fungi against several phytopathogenic isolates as tested using a dual-culture method showed that isolates Tpi_io_1 (Talaromyces pinophilus), Pbi_io_2 (Penicillium bilaiae), Asc_io_1 (Aspergillus sclerotiorum) and Gla_io_1 (Geosmithia lavendula) displayed a broad range of antagonistic activities. Conclusion Fungi associated with C. vestitus had a variable range of pathogenic activity on pistachio stem. Phytopathogenic fungi were antagonized by different fungal isolates which could be promising in pistachio protection against phytopathogenic fungi. Significance and Impact of the Study This study is the first that investigated the diversity of fungi associated with C. vestitus and evaluated both their phytopathogenic activity and antagonistic potential against fungal phytopathogens.