Microbes that can be cultured and degrade petroleum are of particular interest for biotechnology such as bioremediation. This study aims to isolate and identify culturable petroleum-degrading bacteria and fungi from Brunei Darussalam, which has not previously been explored. A total of eight bacterial and nine fungal isolates that could degrade petroleum were obtained from petroleum-contaminated water or soil samples. DNA barcoding using 16S rRNA gene sequence identified five different bacterial genera which were Bacillus, Enterobacter, Micrococcus, Pseudoaltermonas and Pseudomonas. DNA barcoding using rRNA-ITS gene sequence identified nine different fungal taxa which were Aspergillus, Cladosporium, Exophiala, Flavodon, Hypocreales, Nectriaceae, Penicillium, Peniophora and Trichoderma. Biolog provided additional support to the identification of some isolates. This study is the first to report these unique microbes from Brunei Darussalam, which are of ecological and biotechnological value.