This study presents investigations of the periphytic diatoms on artificial (glass slides) and natural substrates in the karstic, limnocrene spring of Bunica situated in the south of Bosnia and Herzegovina. Investigations were performed in summer 2010. Samples were collected every seven days for eight weeks. Physical and chemical characteristics of water, temperature, oxygen saturation, dissolved oxygen, electric conductivity and nutrients as well as fl ow velocity at sample site, were measured simultaneously with each sampling. Physical and chemical characteristics showed low temperature oscillations, good aeration and oligotrophic conditions. In general, greater diatom diversity was noted on natural substrate. A total of 104 diatom species were found on natural substrate and 82 on glass slides. The best represented genera on both types of substrate were Gomphonema and Navicula (each with eight species), Nitzschia (with six species), and Cocconeis (with five species). Achnanthidium exiguum, Achnanthidium minutissimum, Amphora pediculus, Cymbopleura amphicephala and Surirella minuta were recorded in all samples of natural substrate and Gomphonema minutum in artificial substrate samples.