Seasonal droughts in Mediterranean streams shape their physical, chemical, and biological characteristics. Thus, droughts have the potential to alter resources at the base of the food web, which in headwater streams are primarily allochthonous and secondarily autochthonous organic matter (OM). In the present study we assessed the quality of basal resources in a Mediterranean stream during a drought episode before and after a non-flow period (NF). Fatty acids (FA) and sterols were analyzed in the benthic substrata (leaves and sand and cobbles biofilm) and transported OM (particulate and dissolved fractions). FA and sterols were selected as indicators of resource quality because they include essential molecules for consumers and may be used as biomarkers of OM sources. The drying-rewetting process determined a general reduction in the total and essential FA of benthic substrata and transported particulate OM, and a shift from predominantly autochthonous to allochthonous OM. Furthermore, the sterol composition did not change between the drying and rewetting phases and the rewetting did not cause the leaching of FA in dissolved OM. The epilithic biofilm and leaves were the most important sources of essential FA and sterols, while the sand biofilm was the poorest source of these lipids. Our conclusions enhance the understanding of the mechanisms underlying the effects of droughts on basal resource quality in streams.