The increase in global atmospheric mercury circulation, caused by anthropogenic activities, poses a significant risk to public health. Inorganic mercury that is deposited on the landscape moves to lake sediments, where it can undergo methylation by microorganisms. Methylmercury (MeHg) enters lacustrine food systems and bioaccumulates and biomagnifies. The processes that lead to this are complex and include multiple environmental facets that can be modelled using a structural equation model (SEM). However, SEM requires the pre-conceptualization of causal relationships within the model. We assessed how ecological and catchment characteristics influence total mercury (T-Hg) concentrations in brook trout (Salvelinus fontinalis). Brook trout were collected from 31 ponds in western Newfoundland and grouped into four spatial clusters, each exhibiting diverse catchment characteristics. Morphological and physiological traits such as length, mass, age, sex, and isotopic compositions of 15N and 13C of fish were measured. The T-Hg concentration varied from 0.013 to 0.316 mg/kg (fresh weight), with a mean of 0.129 mg/kg, well below the permitted maximum level of 0.5 mg/kg. We found significant differences (p < 0.05) in length-adjusted T-Hg concentrations among sampled ponds and lake clusters. We identified a significant positive relationship (p < 0.05) between T-Hg and principal components that are related to landcover changes from forest to shrubland. These findings allude to the possibility that regional effects, such as landcover and topography, drive the spatial variability of fish T-Hg concentrations. This work also demonstrates the utility of a holistic SEM approach considering direct and indirect pathways of mercury bioaccumulation in fish.