Aims: This study aims to determine the antibacterial potential and identify the bioactive compounds of Hypoxylon monticulosum isolated from marine macroalgae Ulva lactuca. Methodology and results : Ulva lactuca was collected from the Desaru coast, Johor, Malaysia and three endophytes were isolated following surface sterilisation. One fungal isolate was further characterised by the morphology of white, yellowish colonies and fibrous with a waxy structure indicative of a member from the genus Hypoxylon. Molecular identification through internal transcribed spacer (ITS) sequence analysis matches the reference sequence with more than ≥98% homology to Hypoxylon monticulosum AS26-D8. Minimum inhibition concentration (MIC) of the fungal ethyl acetate (EA) extract was determined against five human pathogenic bacteria. Wide spectrum antibacterial activity was noted; with MIC against Escherichia coli was 1.25 ± 0 mg/mL, Bacillus subtilis and Enterobacter faecalis both at 5.00 ± 0 mg/mL, and finally, both Staphylococcus aureus and Klebsiella pneumoniae were 10.00 ± 0 mg/mL, respectively. Bioassay-guided fractionation was performed using solvents of increasing polarities, producing three fractions and analysis by liquid chromatography-mass spectrometry (LC/MS) identified 128 compounds. From these, nine compounds were identified as having biological activities. Dihydrocordoin, D-pantothenoyl-L-cysteine, caffeine and Tumonoic A acid were among the compounds identified as having antibacterial properties. Conclusion, significance and impact of study Hypoxylon monticulosum from marine source has antibacterial potential owing to the compounds previously reported to display antibacterial and other biological properties. The compounds differ from those previously reported in H. monticulosum from terrestrial sources.