Invasive plant species pose a major threat to native flora, in part due to the allelopathic effects of root exudates. The Novel Weapon Hypothesis (NWH) implicates root exudates as a primary factor for successful take over and destruction of native flora by invasive species. However, the precise mechanisms by which invasive species root exudates mediate this impact are unclear. This study aims to compare and evaluate the specific allelochemical impacts of root exudates from the invasive Bidens pilosa on the native fern Pteris multifida under invasive pressure. Specifically, after 10 days’ exposure, label-free proteomics was applied to analyze the proteins and responsive pathway in Pteris multifida Poir. gametophytes upon exposure to two major Bidens pilosa L. root exudates, undecane and palmitic acid. The data show that 2183 proteins were detected in the untreated Pteris multifida gametophyte; 1911 proteins in the gametophyte treated with the undecane, and 2148 proteins in the gametophyte treated with palmitic acid. After exposure for 10 days, undecane treated gametophytes exhibited morphological anomalies and chlorosis; palmitic acid exposure induced no such effects, although development was delayed relative to the control. Using GO functional protein analysis and KEGG pathways detection, we found that the root exudates played different roles on gammopathy growth. Undecane down regulated fatty acid biosynthesis, damaging the cell and chloroplast membrane, and ultimately leading to cell death. Palmitic acid down regulated flavonoid biosynthesis, compromising the gametophyte photosystem and increasing oxidative stress risk. The specific exudate profiles likely facilitate Bidens pilosa invasion, indicating targeted management of key allelochemicals may mitigate invasive impacts on native species.