The present study assesses mercury biomagnification and the trophic structure of the ichthyofauna from the Puruzinho Lake, Brazilian Amazon. In addition to mercury determination, the investigation comprised the calculation of Trophic Magnification Factor (TMF) and Trophic Magnification Slope (TMS), through the measurements of stable isotopes of carbon (δ13C) and nitrogen (δ15N) in fish samples. These assessments were executed in two different scenarios, i.e., considering (1) all fish species or (2) only the resident fish (excluding the migratory species). Bottom litter, superficial sediment and seston were the sources used for generating the trophic position (TP) data used in the calculation of the TMF. Samples from 84 fish were analysed, comprising 13 species, which were categorized into four trophic guilds: iliophagous, planktivorous, omnivorous and piscivorous fish. The δ13C values pointed to the separation of the ichthyofauna into two groups. One group comprised iliophagous and planktivorous species, which are linked to the food chains of phytoplankton and detritus. The other group was composed by omnivorous and piscivorous fish, which are associated to the trophic webs of phytoplankton, bottom litter, detritus, periphyton, as well as to food chains of igapó (blackwater-flooded Amazonian forests). The TP values suggest that the ichthyofauna from the Puruzinho Lake is part of a short food web, with three well-characterized trophic levels. Mercury concentrations and δ13C values point to multiple sources for Hg input and transfer. The similarity in Hg levels and TP values between piscivorous and planktivorous fish suggests a comparable efficiency for the transfer of this metal through pelagic and littoral food chains. Regarding the two abovementioned scenarios, i.e., considering (1) the entire ichthyofauna and (2) only the resident species, the TMF values were 5.25 and 4.49, as well as the TMS values were 0.21 and 0.19, respectively. These findings confirm that Hg biomagnifies through the food web of Puruzinho Lake ichthyofauna. The migratory species did not significantly change mercury biomagnification rate in Puruzinho Lake; however, they may play a relevant role in Hg transport. The biomagnification rate (TMS value) in Puruzinho Lake was higher than the average values for its latitude, being comparable to TMS values of temperate and polar systems (marine and freshwater environments).