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Geographical distribution and abundance of the pupae of six blackfly species of medical and veterinary concern were studied in eastern Spain according to three different sets of explanatory variables including in‐stream variables, both (i) abiotic (i.e., physicochemical) and (ii) biotic (i.e., richness and abundance of either taxonomically or ecologically close related taxa), as well as (iii) meteorological and landscape variables. The results showed specific habitat requirements for pupation in Simulium (Boophthora) erythrocephalum (De Geer, 1776) and Simulium (Wilhelmia) equinum (Linnaeus, 1758), two of the six species studied regarding elevation and temperature. While the rest of the species showed a certain degree of ecological overlap, co‐occurrence was in general low, which suggested that antagonistic biotic factors may be important in structuring blackfly assemblages. In effect, biotic predictors explained a high proportion (50%–70%) of the variability in the abundance of the pupae of the most generalist blackfly species, although further studies are needed to disentangle the sign of interspecific interactions. At the landscape level, S. (W.) equinum and S. (W.) pseudequinum Séguy, 1921 breeding habitats were associated with the presence of pig farms, and S. (Simulium) reptans (Linnaeus, 1758) and S. (B.) erythrocephalum with the presence of cattle. [ABSTRACT FROM AUTHOR]