Peat and various peat preparations have been successfully used in balneology. When considering the biological effects of peat, humic substances have been found to be of particular importance. The content of humic, hymatomelanic and fulvic acids as well as main characteristics of peat were measured in three areas with balneological potential, selected according to previous research and mapping of Estonian peatlands. It appeared that the content of bioactive substances in the sampled peat layers was relatively high. The concentrations were highest in the Parika peatland: humic acids 39.3%, hymatomelanic acids 19.3% and fulvic acids 1.3%. The main factors influencing the levels of bioactive substances were the degree on humification (more humified peat had higher concentrations) and peat type (forest(pine)–cottongrass composition increased the concentration levels). As lipids had high correlation with hymatomelanic acids and trace elements with fulvic acids, the mentioned components could be respectively bound to these humic substances.