Populus species are susceptible to infection by microbial pathogens that severely affect their growth and substantially decrease their economic value. In this study, two pathogenesis-related protein genes consisting of Beauveria bassiana chitinase (Bbchit1) and motherwort lipid-transfer protein (LJAMP2) were introduced into Chinese white poplar (Populus tomentosa Carr.) via Agrobacterium-mediated transformation using the hygromycin (hyg) and neomycin phosphotransferase (NPTII) genes as selectable markers, respectively. Polymerase chain reaction analysis confirmed the stable integration of transgenes in the genome of transgenic plants. In vitro assays showed that inhibitory activity against the fungal pathogen Alternaria alternata (Fr.) Keissler was evident from the crude leaf extracts from transgenic plants. Importantly, the double-transgenic plants exhibited significantly higher resistance to the pathogen than either of the single-gene transformants and wild-type plants when inoculated with A. alternata. The level of disease reduction in double-transgenic lines was between 82 and 95%, whereas that of single-gene transformants carrying either LJAMP2 or Bbchit1 was between 65 and 89%. These results indicated that the combined expression of the LJAMP2 and Bbchit-1 genes could significantly enhance resistance to necrotrophic fungal pathogens in poplar.