Algal and cyanobacterial composition in biofilms is controlled by several factors, including light, nutrients, substratum, hydrodynamics, and grazing. Another potential determinant rarely taken into account is allelopathy, i.e., negative chemical interactions between competitors. We examined the interaction between abiotic variables (light, nutrients, and salt stress) and allelopathy by analyzing the effects of culture conditions on production of allelochemicals by 2 green algal species (Uronema confervicolum and Desmodesmus quadrispina). Production of allelochemicals was greater during the exponential growth phase than during other growth phases and was affected by light, nutrients, and salt concentrations, albeit differently for the 2 species. We then exposed 3 target species (D. quadrispina, Nitzschia palea, and Nostoc PCC 7120) to compounds produced by D. quadrispina, Monoraphidium aff. dybowski, or U. confervicolum. Nutrient-depleted conditions decreased sensitivity, whereas elevated salt concentrations increased sensitivity of D. quadrispina and N. palea to extracts from each of the 3 donor species. Effects of light conditions on sensitivity of target species to donor algal extracts varied among target species. Our results showed that, in benthic biofilms, the magnitude of response to allelochemicals depends on the target species, the donor species, and the abiotic conditions under which they are growing. The role of allelopathy in controlling species composition of biofilms probably is underestimated because the outcome of such complex interactions is difficult to predict.