Karst springs are extremely diverse in terms of morphology, microhabitat structure, hydrology and hydrogeology and consequently unique in their composition of aquatic invertebrate communities. The two studied karst springs of the Gacka River in Croatia, Majerovo and Tonkovića Vrilo, are large limnocrenous springs located in different sub-catchments. They differ in terms of groundwater residence time, discharge regime and self-purification capacity of the water. Invertebrate meiofauna and macrozoobenthos samples were collected seasonally following the modified AQEM protocol to (1) assess the structure and abundance of the benthic assemblages in different microhabitats in relation to ecological and spatial correlations in each spring; (2) assess and compare the habitat preferences of the aquatic invertebrate community; and (3) assess the variation in habitat preferences at the microhabitat level. A total of 70 invertebrate subsamples from dominant inorganic and organic substrates were collected along 100 metres of each spring. Most physico-chemical water parameters differed significantly between the two springs, with values generally lower in Majerovo Vrilo. Primer 6.1 SIMPER analysis identified vegetation and animal taxa that were most responsible for the multivariate distance between seasonal samples and showed that isopods and amphipods were most abundant in both springs during all sampling periods. Invertebrate species richness was significantly higher in Tonkovića Vrilo, where the higher concentrations of dissolved ions were measured. Non-metric multidimensional scaling (nMDS) of ordination plot revealed a separation of invertebrate assemblages between springs at the microhabitat level. The classification of vegetation-animal assemblages into types based on microhabitats is intended to characterize the life requirements of each recorded species. This study has shown the importance of overlaying multifunctional ecological data to assess land-use changes in different sub-catchments of two neighbouring springs.