The accidental release of Hazardous and Noxious Substances (HNS) can negatively affect the environment and human health. Biofuels are promising alternatives to fossil fuels as they are produced from renewable sources (i.e., vegetable oils or animal fats). However, biofuels are considered one of the HNS categories that are frequently transported and will impact the ecosystem if spilled on water. Although studies support the biodegradation of some commercially available biodiesels in water, the non-volatile nature of newly developed alternative biofuels (ABFs), such as biomass-derived pyrolysis oils, and their unique physicochemical properties suggest that these oils may behave differently if spilled in water. This study aimed to determine the fate and transformation (chemical or biological) of pyrolysis oils in Canadian marine waters through bench-scale biodegradation experiments. The biodegradation experiments were performed by spilling pyrolysis oils (100 µL), obtained from two different suppliers, in 200 mL of water, collected from the Canadian East Coast, Pacific Ocean, and the Arctic Ocean, under aerobic conditions (200 rpm) at 4 °C water for 21 days. Samples were collected at T3 (three hours post-spill) and the end of the test and analysed for total organic carbon content (TOC) and microbial community analysis by 16S rRNA gene sequencing. Visual observation of the bio-oil showed that the oil sank to the bottom of the water, but the wood oil floated first and sank after a short period of time. Both oils experienced limited biodegradation (<20%) in the marine waters. Further analysis of oil properties in waters will unfold the factors that led to the low biodegradation of the tested oils in cooler water.