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Ambrosia beetles bore into host trees, and live with fungi symbiotically that serve as a food source. However, it is challenging to directly observe these beetles in the wild. In this study, <i>Euwallacea interjectus</i> (Blandford) (Coleoptera: Curculionidae: Scolytinae), a pest of fig trees in Japan, were reared under artificial conditions to emulate the behavior of ambrosia beetle. Fungi were isolated from the adult females of <i>E. interjectus</i> to identify the species associated with secondary symbiosis. In total, nine filamentous fungi and one yeast were identified using morphological characteristics and DNA sequence data. <i>Neocosmospora metavorans</i> (Hypocreales: Nectriaceae), <i>Fusarium</i> sp. (Hypocreales: Nectriaceae), that is undescribed, and <i>Meyerozyma guilliermondii</i> (Saccharomycetes: Saccharomycetales) (yeast) were isolated more frequently from the head (including from mycangia, the fungus-carrying organ) than from the thorax and abdomen of adult beetles. <i>Neocosmospora metavorans</i> was the dominant species isolated from 12 out of 16 heads at 200 to 3300 CFUs/head, compared to the primary mycangia fungus from wild beetles, i.e., <i>Fusarium kuroshium</i> (Hypocreales: Nectriaceae). Temperature had a marked effect on fungal growth in the three symbiont species. Our results represent a major paradigm shift in understanding beetle–fungal interactions, as they show specific symbiont switching can occur in different nesting places.