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WRKY transcription factors (WRKY TFs) are one of the largest protein families in plants, and most of them play vital roles in response to biotic and abiotic stresses by regulating related signaling pathways. In this study, we isolated two WRKY TF genes <i>PtrWRKY18</i> and <i>PtrWRKY35</i> from <i>Populus</i><i>trichocarpa</i> and overexpressed them in Arabidopsis. Expression pattern analyses showed that <i>PtrWRKY18</i> and <i>PtrWRKY35</i> respond to salicylic acid (SA), methyl JA (MeJA), abscisic acid (ABA), <i>B. cinereal</i>, and <i>P. syringae</i> treatment. The transgenic plants conferred higher <i>B. cinerea</i> tolerance than wild-type (WT) plants, and real-time quantitative (qRT)-PCR assays showed that <i>PR3</i> and <i>PDF1.2</i> had higher expression levels in transgenic plants, which was consistent with their tolerance to <i>B. cinereal.</i> The transgenic plants showed lower <i>P. syringae</i> tolerance than WT plants, and qRT-PCR analysis (<i>PR1</i>, <i>PR2</i>, and <i>NPR1</i>) also corresponded to this phenotype. Germination rate and root analysis showed that the transgenic plants are less sensitive to ABA, which leads to the reduced tolerance to osmotic stress and the increase of the death ratio and stomatal aperture. Compared with WT plants, a series of ABA-related genes <i>(RD29A</i>, <i>ABO3</i>, <i>ABI4</i>, <i>ABI5</i>, and <i>DREB1A</i>) were significantly down-regulated in <i>PtrWRKY18</i> and <i>PtrWRKY35</i> overexpression plants. All of these results demonstrated that the two WRKY TFs are multifunctional transcription factors in plant resistance.