A growing interest in the importance of simuliids in health and animal welfare in certain areas in Europe, such as around the Mediterranean basin, together with the fact that there is fragmentary ecological information on the pre-imaginal stages of these dipterans has led to the need to carry out further research. We studied the abundance and distribution of pre-imaginal stages—mainly the pupae—of simuliid species, and examined their assemblages in relation to environmental gradients identified in Mediterranean basins of eastern Spain. We report a total of 21 black fly species from the set of studied streams, where largely the most common species were Simulium (Simulium) intermedium and Simulium (Simulium) ornatum, two species of medical and veterinary concern. We found that pre-imaginal simuliid assemblages were strongly related to elevation, water temperature and turbidity, dissolved oxygen concentration and water velocity. The analysis of the optima and tolerances for these environmental factors suggests differences in ecological requirements of the pre-imaginal stages of the reported simuliid species. Likewise, it was found that the pupae of some species were strongly associated with specific substrates. We hope that our research results on autoecology of black fly species will contribute to the improvement of pest management programs.