Black flies (Simuliidae) are a very significant component of lotic habitats and they also have important medical and veterinary implications because adult females of most species are hematophagous. This study was carried out in Plitvice Lakes National Park, on four sites: Spring of Bijela rijeka, Upper reach of Bijela rijeka, Tufa Barrier Labudovac and Tufa Barrier Kozjak-Milanovac. Samples were collected once a month using pyramid-type emergence traps, over a period from 2007 until the end of 2010. The main aims were: to identify the main ecological factors affecting the species composition and abundance of black flies, to determine changes in phenology patterns, to determine the longitudinal zonation from Spring of Bijela rijeka to downstream, and to determine differences in the number of black flies on certain microhabitats. During the study, a total caught were 39187 individuals. Temperature, water discharge and type of habitats are the factors that had the most influence on the black fly assemblages. It was found that black flies do not favor sites close to springs, in contrast, they were found in greater numbers at tufa barriers. The highest diversity has been noted at Tufa Barrier Labudovac and the highest number of individuals at Tufa Barrier Kozjak-Milanovac. Two species were identified at sites close to springs: Simulium (Nevermannia) costatum and Simulium (Odagmia) monticola. Specie Simulium (Nevermannia) costatum was found at all sites, but in different proportions. On the other hand, at tufa barriers Simulium (Eusimulium) angustipes remained the most abundant species, which is very important because it is a vector in transmitting avian trypanosomes.