Display omitted •Hexadecyl-methylimidazolium chloride was effective in inhibiting growth and biofilm formation by Navicula sp.•Activity of ionic liquid was dependent on the alkyl side chain length.•Ionic liquid disrupted cell membrane and caused leakage of intracellular components in diatom cells.•Natural phototrophic biofilms were prevented with ≤5μM of ionic liquid. Biofilm formation is problematic and hence undesirable in medical and industrial settings. In addition to bacteria, phototrophic organisms are an integral component of biofilms that develop on surfaces immersed in natural waters. 1-Alkyl-3-methyl imidazolium ionic liquids (IL) with varying alkyl chain length were evaluated for their influence on the formation of monospecies (Navicula sp.) and multispecies biofilms under phototrophic conditions. An IL with a long alkyl side chain, 1-hexadecyl-3-methylimidaazolium chloride (C16(MIM)Cl) retarded growth, adhesion and biofilm formation of Navicula sp. at concentrations as low as 5μM. Interestingly, C16(MIM)Cl was very effective in preventing multispecies phototrophic biofilms on fibre reinforced plastic surfaces immersed in natural waters (fresh and seawater). SYTOX® Green staining and chlorophyll leakage assay confirmed that the biocidal activity of the IL was exerted through cell membrane disruption. The data show that C16(MIM)Cl is a potent inhibitor of phototrophic biofilms at micromolar concentrations and a promising agent for biofilm control in re-circulating cooling water systems. This is the first report that ionic liquids inhibit biofilm formation by phototrophic organisms which are important members of biofilms in streams and cooling towers.