Mango (Mangifera indica) is an economically significant crop, and is affected by dieback in nearly all commercial production areas. Due to the wide range of organisms previously associated with these disease symptoms in Florida, isolations and pathogenicity tests were carried out to determine the causal organism. The pathogen was identified as Neofusicoccum batangarum based on genetic sequences from three loci (internal transcribed spacer of the rDNA (ITS), β-tubulin (BT), and translation elongation factor 1-α (EF)), recommended for members of the Botryosphaeriaceae family. Possible infection routes were determined by inoculating wounded and unwounded stems with N. batangarum. Trees wounded prior to pathogen inoculation developed larger lesions (5.85 cm ± 1.51) than unwounded trees (0.51 cm ± 0.48), p < 0.0003. In addition, lesions only developed at a small number of inoculation sites in the absence of wounds (14.3%), compared to 93% when stems were wounded. No necrosis was observed in the negative controls. This study provides molecular data on N. batangarum, and evidence of its role causing mango dieback in Florida.