The World Health Organization (WHO) highlights on the antimicrobial resistance (AMR) in bacterial pathogens as a leading cause fatalities and health risks associated with bacterial infections in humans. As a result, new and effective treatment options are urgently needed. Secondary metabolites (SMs) from microorganisms such as bacteria and fungi are the major source for the currently used antibiotics. Fungi belonging to the Ophiostomatales have not been studied much with regards to the production of SMs. In this study, around 100 different strains of Leptographium spp., belonging to the order Ophiostomatales, were screened for bioactivity via agar plug assays. From the bioactivity screen, several strains of Leptographium wingfieldii showed an indication of antimicrobial activity especially against an efflux deficient strain of Pseudomonas aeruginosa PAO750. SMs were recovered from spent media (solid state fermentation) using the ethyl-acetate based extraction protocol; the “crude extracts” were prepared by evaporation of the solvent. Chemical compounds produced by these fungi were characterized using liquid chromatography-mass spectrometry (LC-MS). Antibase 2012 was used to identify novel compounds. Previously known compounds with antimicrobial activity such as Limazepine B1 and B2, and Pulcherriminic acid were identified. However, some compounds yielded signals that were “no hits” in the Antibase search. These molecules potentially could have novel chemical or biological significance. In conclusion, the detection of SMs from Leptographium spp. may lead to the discovery of new antibiotics and help in treatments against antibiotic resistant bacteria.