Several species in the Botryosphaeriaceae family cause wood stain, cankers, and dieback of trunks and branches in a wide range of forest tree species. The aim of this study was to characterize the botryosphaeriaceous fungi associated with decline symptoms observed in Acacia mangium and Pinus caribaea var. hondurensis, two economically important forest tree species grown in commercial plantations in Venezuela. Fungi isolated from symptomatic samples collected from both hosts in commercial sites were identified based on their morphology and DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) and part of the β-tubulin and translation elongation factor 1-α genes. Lasiodiplodia theobromae and L. venezuelensis were routinely isolated from A. mangium and P. caribaea var. hondurensis. Additionally, the novel species Diplodia guayanensis was isolated and characterized from symptomatic and asymptomatic tissues of A. mangium. Multigene phylogenetic analyses along with restriction fragment length polymorphism studies further supported the identification of these species. A pathogenicity study was conducted under natural conditions and 12 weeks after inoculation all Botryosphaeriaceae spp. were shown to be highly virulent on A. mangium. Contrary, no lesions were observed in the wood of P. caribaea var. hondurensis when inoculated with L. theobromae and L. venezuelensis. However, both species were consistently reisolated from the asymptomatic tissue beyond the inoculation point. This study contributes to a better understand the role that species in the Botryosphaeriaceae play on disease symptoms and dieback of A. mangium and P. caribaea var. hondurensis from plantations in eastern Venezuela.