The archaeological investigations of Delos’s latrines unveiled a surprising discovery: the remains of freshwater fish (Cyprinidae, Sander lucioperca, Siluris glanis). The bones, retrieved in contexts dating back to the Hellenistic period (2nd–1st centuries BCE), raise the question of the origin of the fish and their significance within an archipelago situated in the heart of the Aegean Sea. Despite Delos lacking permanent natural freshwater sources or reservoirs, a convergence of archaeological, archaeozoological, and epigraphic evidence indicates that these fish were deliberately transported while alive and subsequently held within an artificial lake under the administration of the Apollonian sanctuary. Stable isotope analysis was considered to gain insight into the origin(s) and the environment in which the fish were raised. Given the destructive nature of the method and the rarity of the bones, every effort was made to conserve as much information as possible before analysis. Micro-CT scans were produced for all bones, along with 3D prints of samples that were subsequently destroyed. Micro samples were taken for FTIR-ATR analysis both to estimate collagen content and to gauge the effect of diagenesis on bone carbonates. Samples failing to meet quality standards were omitted from the analysis. Analysis revealed high alteration of carbonates. However, despite the particularly harsh environmental conditions of Delos, collagen conservation was promising as all except 1 bone (24 bones analyzed) were estimated to provide enough collagen for extraction. Carbon and nitrogen stable isotope analysis of bone collagen was then carried out. High variability in the data seems to indicate multiple origins for the fish. Indeed, the results spread further than expected considering both species-specific diets and intraspecies trophic levels (based on size estimations). High δ13C values compared to two marine fish bones also collected on Delos suggest a slightly brackish environment for some samples. Regionally, little data has been produced for archaeological freshwater fish, significantly complicating comparisons and interpretation. This study's results do nonetheless fall within the range of the data produced for prehistoric freshwater fish dating from prehistoric times in continental north Greece. These isotopic data solidify the hypotheses that freshwater fish were transported live and stocked in the sacred lake of Delos, possibly sometimes for several years if the pluviometry permitted.