A predominance of alewives (Alosa pseudoharengus), a species having high thiaminase activity, in Lake Ontario lake trout (Salvelinus namaycush) diets, has been related to thiamine deficiency in lake trout eggs during 1994–2004. The late 1990s invasion by round goby (Neogobius melanostomus), that appear to have thiaminase activity of low biological activity, represented a potential to reduce the dietary importance of alewife and, as a result, increase lake trout thiamine levels if they became sufficiently important in lake trout diets. To evaluate whether lake trout thiamine levels increased as alewives were displaced by round gobies in lake trout diets, we collected 199 lake trout ranging from 305 to 893 mm in 2005–2006 and measured their muscle thiamine levels and diet composition. Diet composition (percent by weight) was estimated from MixSIR based on stable isotopes (δ¹⁵N and δ¹³C) measured from lake trout and their prey. Overall, alewife and goby dominated lake trout diet (78%), with round goby dominating the diet (55–57%) of smaller individuals (<600 mm), and alewife dominating the diet (59–73%) of larger, reproductively active individuals. Lake trout muscle thiamine declined with increases in lake trout length and the proportion of alewife eaten (p < 0.01). The proportion of lake trout below 500 pmol/g thiamine also declined; this threshold is associated with a loss of equilibrium in adults. Despite the increasing albeit size-related consumption of round goby, it remains inadequate as muscle thiamine levels in mature lake trout (i.e., >600 mm) during 2005–2006 appear unchanged from levels observed in 1996.