These datasets support the data provided directly in the scientific publication titled "Tomato Leaf Curl New Delhi Virus Spain Strain Is Not Transmitted by Trialeurodes vaporariorum and Is Inefficiently Transmitted by Bemisia tabaci Mediterranean between Zucchini and the Wild Cucurbit Ecballium elaterium", published in the Insects journal. The figure included in the datasets highlights agroinoculation experiments with tomato leaf curl New Delhi virus Spain strain using different source plant–target plant–whitefly species combinations. ToLCNDV (DNA-A and DNA-B) was detected by molecular hybridization after tissue printing on nylon membranes using specific digoxigenin-labelled DNA probes for each viral component. (A) Prints of zucchini plants inoculated using Trialeurodes vaporariorum (T. v.) or Bemisia tabaci MED (B. t.) as a vector and infected zucchini plants as the virus source. (B) Prints of Ecballium elaterium plants using B. t. as a vector and infected zucchini plants as the virus source. (C) Prints of zucchini plants using B. t. as a vector and infected E. elaterium plants as the virus source. Blots from one of the two independent experiments (Experiment 1 in the Table) carried out for each source plant–target plant–whitefly species combination are shown. Prints of ToLCNDV-infected zucchini plants included as positive controls are outlined with red squares. The table included in the datasets show the results of transmission experiments of tomato leaf curl New Delhi virus Spain strain between zucchini cv. Milenio and Ecballium elaterium plants by Trialeurodes vaporariorum and Bemisia tabaci MED. Infection was determined by tissue-printing and molecular hybridization with digoxigenin-labelled ToLCNDV DNA-A and DNA-B probes. Blots from experiment (Exp.) 1 of each source plant–target plant–whitefly species combination are shown in the Figure. The provided datasets are further discussed and interpreted in detail, as well as their subsequent results, in the scientific publication. This research was conducted within the VIRTIGATION project, which is part of the EU Open Research Data pilot. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101000570.