Globally, both natural water bodies and aquaculture systems are being severely contaminated by heavy metals due to rising anthropogenic activities. Fish living in aquatic environments can easily accumulate metals in their bodies, which can then be transferred to consumers and put them at risk. In this study, metal concentrations (Pb, Cd, Cr, As, Mn, Cu, Zn) in different organs (gill, liver, and muscle) of farmed and wild Barramundi (Lates calcarifer) fish from the northern Bay of Bengal were evaluated to quantify and compare contamination levels and related human health risk. Heavy metal concentrations were higher in liver tissues of farmed Barramundi than in wild Barramundi, with the following relative mean values in the liver, gills, and muscle: Zn > Cu > Pb > Mn > Cd > Cr > As; Zn > Cr > Cu > Pb > Mn > Cd > As; Zn > Pb > Cu > Cr > Mn > Cd > As; Zn > Pb > Cu > Cr > Mn > Cd > As; and Zn > Pb > Cu > Cr > Mn > Cd > As, respectively. The differences in heavy metal accumulation observed between farmed and wild fish were probably related to the differences in their environmental conditions and dietary element concentrations. However, ANOVA indicated that the variation of metals in wild and Barramundi was not statically significant. Pb concentrations in the liver tissue of farmed Barramundi exceeded the national and international threshold limits, whereas concentrations of other metals were within the limit. Among the examined organs in both fish species (wild and farmed), muscle had the lowest concentration compared to others, and liver was the target organ for Pb, Cu, and Cd accumulations. Metals such as Zn and Mn exhibited higher concentration in the gills. However, all the studied heavy metals were below the maximum permissible limits of national and international standards, but the mean concentrations of Pb and Cd values in the liver of farmed Barramundi exceeded all international and national guidelines. Based on the contamination factors (CF) and pollution indices (PLI and MPI), the degree of contamination in the fish organs was as follows: gills > liver > muscle. The major accumulation tissues for both farmed and wild fish were found to be the gills (MPI = 0.970) and the liver (MPI = 0.692). Based on the estimated daily intake (EDI), the fish samples examined in this study are safe for human consumption as within the recommended daily allowance (RDA) range established by various authorities. According to the Target Hazard Quotient (THQ) and Carcinogenic Risk (CR) calculations, though the Barramundi fishes depicted no potential hazard to humans, farmed fish posed a higher health risk than wild fish.