AbstractRecent observations in marine ecosystems show that the presence of thiamine regulates primary production, but little is known about the ecological effect of thiamine in streams. We conducted nutrient enrichment experiments in four streams in the New York's Adirondack Mountains using nutrient diffusing substrates to evaluate the influence of thiamine (vitamin B1) on the growth of stream periphyton. Contrasting treatments in our study included nutrient additions of thiamine (C12H17ClN4OS·HCl), nitrogen (NH4Cl), and phosphorus (NaH2PO4). Thiamine limitation occurred in 12 of 14 experiments conducted from June through October in 2015–2017, nitrogen limitation occurred in eight experiments, and phosphorus limitation in one experiment. The magnitude of response of periphyton to thiamine enrichment varied among seasons, years, and streams. The growth‐enhancing effect on periphyton biomass from thiamine or nitrogen addition typically occurred between 8 and 32 d of incubation, though the periphyton accumulation rate declined after 16–24 d of incubation. Our results showed that thiamine is a limiting nutrient for primary producers in our study streams and its effect size is similar to that of nitrogen. These findings extend the prior recognition of thiamine's substantial influence on marine photosynthetic eukaryotes to having a similar role in freshwater ecosystems.