Dragon fruit (Hylocereus polyrhizus) is an economically promising fruit in Bangladesh. The cultivation of dragon fruit has increased fourfold within a decade due to its popularity. Recently, a new disease known as stem canker was reported in some plantations of dragon fruit in Bangladesh, which forced some farmers to abandon their cultivation. This study aimed to explore the morphological, molecular, and cultural characteristics as well as host range of the causal agent associated with this destructive disease. Morphologically similar eight fungal isolates were recovered from eight canker symptomatic dragon fruit stems. Among them, two isolates (namely BU-DLa 01 and BU-DLa 02) were used for a detailed study. Morphological parameters and phylogeny of sequence data of internal transcribed spacer (ITS1, 5.8S rRNA, and ITS2), β-tubulin, and translation elongation factor 1-α identified the isolates as Lasiodiplodia theobromae. The cultural features were studied hinged on the growth of the two isolates on various media, temperature, and pH. Though the mycelial growth of the fungi was supported by all the media tested, potato dextrose agar was the most suitable one for both isolates. The fungi thrived well at a temperature of 25-35°C and 5.5-6.5 pH. Inoculation trials of dragon fruit stem ascertained Koch's postulate. In host range test, the isolates were found pathogenic toward mango, guava, banana, and the fruits of dragon fruit. These data will contribute not only to understanding the biology of L. theobromae as a newly recognized pathogen of H. polyrhizus but also will help in designing a proper management package against this pathogen.