Evaluation of the prevalence of virus and viroid infections was conducted in a grapevine field collection in Valencia, Spain. Samples of autochthonous and traditional grapevine cultivars were collected during November 2011 and tested for the presence of fourteen viruses and five viroids, using RT-PCR. The prevalent viruses were Grapevine rupestris stem pitting-associated virus (GRSPaV: 49% infected samples) and Grapevine leafroll-associated virus 2 (GLRaV-2: 15% of samples). GLRaV-1, GLRaV-3, GLRaV-4 (variants 4 and 5), Grapevine fanleaf virus, Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) and Grapevine virus A were also detected. Hop stunt viroid (HSVd: 92% of plants infected) and Grapevine yellow speckle viroid 1 (6% of plants) were also detected. Mixed infections with two, and up to six different viruses and/or viroids were common. Only five samples (4%) were free from 19 pathogens tested. This is the first report of GLRaV-4 (variants 4 and 5) in the Valencia region of Spain, and the first record of GRSPaV and GRVFV in this country. Phylogenetic analyses performed with the sequences of these viruses showed that the Spanish isolates of GLRaV-4, GFkV and HSVd belong to new phylogenetic groups.