In the science of drug discovery, ultraviolet (UV) irradiation has been applied to induce mutagenesis in fungi to provide possibilities for the stimulation or enhancement of fungal biosynthetic capabilities. This study was carried out to evaluate the effect of UV radiation on the biosynthesis of antibacterial secondary metabolites in an endophytic Lasiodiplodia theobromae. Using standard methods, the fungus was isolated from healthy leaves of Cola acuminata and identified based on PCR amplification and genomic sequencing of the internal transcribed spacer (ITS) region. Cultures of L. theobromae were exposed to UV radiation at different time intervals of 1, 2 and 5 min. The fungus was subjected to solid-state fermentation in rice medium before and after UV treatments. The fungal secondary metabolites were extracted and tested for antibacterial activity using the agar diffusion method. Compounds present in the obtained extracts were identified by HPLC and GC-MS analysis. At a concentration of 1 mg/ml, the extract of the wild type L. theobromae (untreated) was observed to only inhibit Staphylococcus aureus, with an IZD of 12 mm. However, the extract of UV-treated L. theobromae (2 min) inhibited S. aureus, Escherichia coli and Pseudomonas aeruginosa with an IZD of 10 and 4 mm respectively. A wide array of compounds in the phenolics, fatty acids, alkaloids and alkanes classes were identified in the UV-treated and untreated fungal extracts. Overall, UV treatments of L. theobromae stimulated the production of seventeen (17) new compounds that were not detected in the untreated strain. The study confirms UV irradiation as an effective method for stimulating microbial biosynthesis of new bioactive compounds, indicating a promising and potentially abundant source of new drug compounds from microorganisms. © Articles by the authors.