The tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (genus Begomovirus, family Geminiviridae) that was originally isolated in India, but has become a serious threat to various horticultural crops in the Indian subcontinent, Southeast Asia, East Asia, Middle East, and western Mediterranean Basin. In Southeast Asia, East Asia, and the western Mediterranean Basin, ToLCNDV has caused huge economic damage to cucurbit crops (cucumber, melon, squash, and zucchini) for which commercial ToLCNDV-resistant cultivars are currently unavailable. In this study, full-length ToLCNDV viral sequences were isolated from cucumber, melon, and zucchini plants exhibiting yellow leaf curl disease in Almeria, Spain. Phylogenetic analysis showed that the isolated viruses consisted of the ToLCNDV-ES strain and strains that were genetically distant from ToLCNDV-[BACu-20] in Southeast Asia, which were previously isolated in the Aceh province of Indonesia. Infectious clones of ToLCNDV-[ES-Alm-Cuc-16] from Spain and of ToLCNDV-[BACu-20] from Indonesia were prepared, and ToLCNDV isolates were agroinoculated to Nicotiana benthamiana, tomato, and cucurbit crops (cucumber, melon, and zucchini). Infection rates were nearly 100% for all inoculated plant species, and disease symptoms were consistently more severe in plants inoculated with ToLCNDV-[BACu-20] compared to plants inoculated with ToLCNDV-[ES-Alm-Cuc-16]. For tomatoes inoculated with ToLCNDV-[ES-Alm-Cuc-16], infection occurred without symptoms in most plants, and only a few plants showed slight vein-yellowing symptoms. In contrast, ToLCNDV-[BACu-20] infection induced typical begomoviral disease symptoms of yellowing and curling of leaves in the same tomato cultivar. The present study highlights the importance of screening genetic resources for ToLCNDV resistance by using ToLCNDV isolates from the intended production area; furthermore, the agroinoculation method used in this study will facilitate breeding resistance against both the Southeast Asian and Mediterranean ToLCNDV isolates.