Mercury (Hg) is a ubiquitous and pervasive environmental contaminant with detrimental effects on wildlife, which originates from both natural and anthropogenic sources. Its distribution within ecosystems is influenced by various biogeochemical processes, making it crucial to elucidate the factors driving this variability. To explore these factors, we employed an innovative method to use northern gannets (Morus bassanus) as biological samplers of regurgitated fish in the Gulf of St. Lawrence. We assessed fish total Hg (THg) concentrations in relation to their geographical catch location as well as to pertinent biotic and anthropogenic factors. In small fish species, trophic position, calculated from compound-specific stable nitrogen isotopes in amino acids, emerged as the most influential predictor of THg concentrations. For large fish species, THg concentrations were best explained by δ13C, indicating higher concentrations in inshore habitats. No anthropogenic factors, such as pollution, shipping traffic, or coastal development, were significantly related to THg concentrations in fish. Moreover, previously published THg data in mussels sampled nearby were positively linked with THg concentrations in gannet prey, suggesting consistent mercury distribution across trophic levels in the Gulf of St. Lawrence. Our findings point to habitat-dependent variability in THg concentrations across multiple trophic levels. Our study could have many potential uses in the future, including the identification of vulnerability hotspots for fish populations and their predators, or assessing risk factors for seabirds themselves by using biologically relevant prey.