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, Euwallacea interjectus 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 E. interjectus 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. Neocosmospora metavorans (Hypocreales: Nectriaceae), Fusarium sp. (Hypocreales: Nectriaceae), that is undescribed, and Meyerozyma guilliermondii (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. Neocosmospora metavorans 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., Fusarium kuroshium (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.