The abundance and predominant groups of bacteria, filamentous fungi and yeasts have been studied by culture-dependent microbiological methods in peat probes obtained in two Latvian balneotherapy spa sites, Kemeri and Baldone. Unsterilized peat samples from both the sites contained 5.7–8.1 log bacterial colony-forming units (CFU) and 3.0–5.3 log fungal CFU per gram of dry peat. Isolated species belonged to Alpha-, Beta-, and Gamma-Proteobacteria, Actinobacteria, Clostridia, Bacilli and Flavobacteria as well as to filamentous fungi and yeasts. The composition of microbial population of the peat from both sites shared just four micro-organism groups (Bacillus mycoides, Burkholderia cepacia, Streptomyces spp. and Trichoderma spp.) within totally 36 groups identified. No pathogenic bacteria or fungi and no faecal pollution indicators were recovered. Decimal reduction doses for micro-organisms in peat samples and radiation sterilization doses of peat for the gamma and electron beam radiation were determined. The highest radiation resistance was observed for B. mycoides and Aureobasidium sp. Gamma-sitosterol was the most abundant hydrophobic organic compound in both peats according to GC–MS data. All the sterilization procedures increased concentration of alkanes, alcohols, and ketones and decreased the amount of fatty acids. Heat sterilization proved to be more preserving for the peat sterols than the radiation sterilization. It is concluded that the heat and radiation sterilization methods induce different changes of the profile of hydrophobic organic compounds of balneological peats, what may lead to different therapeutic effects at their application.