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<i>Phallus rubrovolvatus</i> is a unique mushroom used for medicinal and dietary purposes in China. In recent years, however, the rot disease of <i>P. rubrovolvatus</i> has seriously affected its yield and quality, becoming an economically important threat. In this study, samples of symptomatic tissues were collected, isolated, and identified from five major <i>P. rubrovolvatus</i> production regions in Guizhou Province, China. Based on combined analyses of phylogenies (ITS and EF1-<i>α</i>), morphological characteristics and Koch’s postulates, <i>Trichoderma koningiopsis</i> and <i>Trichoderma koningii</i> were identified as the pathogenic fungal species. Among these, <i>T. koningii</i> exhibited stronger pathogenicity than the other strains; thus, <i>T. koningii</i> was used as the test strain in the follow-up experiments. Upon co-culturing <i>T. koningii</i> with <i>P. rubrovolvatus</i>, the hyphae of the two species were intertwined, and the color of the <i>P. rubrovolvatus</i> hyphae changed from white to red. Moreover, <i>T. koningii</i> hyphae were wrapped around <i>P. rubrovolvatus</i> hyphae, leading to their shortening and convolution and ultimately inhibiting their growth due to wrinkling; <i>T. koningii</i> penetrated the entire basidiocarp tissue of <i>P. rubrovolvatus</i>, causing serious damage to the host basidiocarp cells. Further analyses revealed that <i>T. koningii</i> infection resulted in the swelling of basidiocarps and significantly enhanced the activity of defense-related enzymes, such as malondialdehyde, manganese peroxidase, and polyphenol oxidase. These findings offer theoretical support for further research on the infection mechanisms of pathogenic fungi and the prevention of diseases caused by them.