Microcoleus is a benthic mat-forming cyanobacterium proliferating in flowing waters globally and able to produce the potent neurotoxin, anatoxin-a, responsible for animal poisonings. The drivers of Microcoleus proliferations and toxin production are little known. Thiamine (vitamin B1) is an essential vitamin for living organisms and the current understanding is that the non-toxic Microcoleus species can produce thiamine, while the toxic strains rely on external sources to obtain it. In this study, we explored the effects of thiamine supplementation on Microcoleus growth and toxin-production using toxic and non-toxic unialgal, non-axenic strains of Microcoleus originating from the same stream reach in South Fork Eel River, California. Both strains were grown for 42 days in BG11 thiamine-free medium (control) or BG11 medium supplemented with 0.1 mg/L of thiamine. Cell density and biovolume, chlorophyll a, and anatoxin concentrations were measured in triplicate in a time-series along the growth cycle on days 3, 7, 10, 14, 17, 21, 29, 35, 42. Thiamine supplementation had no effect on the predicted maximum growth rate of the toxic strain as indicated by the Gompertz model, whereas it was associated with a slower maximum growth rate of the non-toxic strain. The toxic strain produced mainly anatoxin-a (ATX) and lower concentrations of dihydroanatoxin-a (dhATX). Microcoleus cells supplemented with thiamine had higher maximum ATX quota with delayed peak concentrations at day 7, which may provide evidence that thiamine could help stimulate toxin-production. Metagenomic data will elucidate Microcoleus species identity and presence of potential thiamine-producers in cyanobacterial microbiome which may influence the obtained results.