Using different bioinformatics tools to analyze data from previous studies of Verticillium nonalfalfae interaction with hop, we predicted that the V. nonalfalfae secretome is rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling. It includes 263 candidate secreted effector proteins (CSEPs), among which several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants (Avr-Pita1 and MgSM1), while the majority of CSEPs were non-annotated. 44 top priority candidates were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.