Lampreys have a complex life cycle which includes a multi‐year infaunal larval stage (ammocoete). Gut content analysis has generally identified detritus (i.e., unidentifiable organic matter) as the major dietary component to ammocoetes, though algae can also be important. However, gut content preserves only a snapshot of the animal's diet and does not reflect assimilated material. In order to better characterise the nutritional sources supporting ammocoete growth, we analysed ammocoete body tissue and potential dietary sources at two streams using natural Δ14C and δ15N to estimate time‐integrated nutritional support. Bayesian isotope mixing models revealed differences in the importance of sources supporting ammocoetes between sites. Ammocoetes from a stream in a mixed land usage area (~50% agriculture, ~40% forest and ~10% developed) were primarily supported (mean: ~50%) by fresh terrestrial organic matter but were also supported by substantial contributions (mean: ~30%) by aged organic matter (AOM) and autochthonous material (algae; mean ~20%). In a predominantly forested (~90%) headwater stream, different modelling scenarios (uninformed or informed priors) suggested that algal support of ammocoete nutrition ranged from 7% to 45%. However, the model relying on informed priors developed from gut content analysis produced the low estimates, suggesting these were more reliable. When algae were a minor component of the nutrition at the forested site, ammocoetes were highly dependent on AOM (83 ± 26%; mean ± SD). Based on these findings, ammocoete growth and development are predicted to be strongly influenced by both land use and the availability of allochthonous and autochthonous materials of varying ages within streams.