Phallus rubrovolvatusis a unique mushroom used for medicinal and dietary purposes in China. In recent years, however, the rot disease ofP. rubrovolvatushas 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 majorP. rubrovolvatusproduction regions in Guizhou Province, China. Based on combined analyses of phylogenies (ITS and EF1-α), morphological characteristics and Koch’s postulates,Trichoderma koningiopsisandTrichoderma koningiiwere identified as the pathogenic fungal species. Among these,T. koningiiexhibited stronger pathogenicity than the other strains; thus,T. koningiiwas used as the test strain in the follow-up experiments. Upon co-culturingT. koningiiwithP. rubrovolvatus, the hyphae of the two species were intertwined, and the color of theP. rubrovolvatushyphae changed from white to red. Moreover,T. koningiihyphae were wrapped aroundP. rubrovolvatushyphae, leading to their shortening and convolution and ultimately inhibiting their growth due to wrinkling;T. koningiipenetrated the entire basidiocarp tissue ofP. rubrovolvatus, causing serious damage to the host basidiocarp cells. Further analyses revealed thatT. koningiiinfection 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.