Spoilage of chocolate pralines, due to growth of microorganisms tolerating low water activity, causes problems in the confectionary industry. Therefore, an increased knowledge on which organisms are present in the chocolate fillings and their tolerance towards low a_w, pH, ethanol and other preservatives is needed. Using media containing 40-50% glucose (a_w 0.872-0.925) bacteria, yeasts and moulds were isolated from chocolate pralines (a_w 0.70-0.898) of nine manufactures and sugar syrups (a_w 0.854) used as ingredient in chocolate praline production by one of the manufacturers. Isolates were identified by conventional microbiological analyses and by sequencing of their 16S rRNA, 26S rRNA (D1/D2-region) or calmodulin genes. Further, for several species the identity was confirmed by amplification and sequencing of additional genes. In total 677 isolates were identified as belonging to ten different bacteria species, six yeast species and ten mould species with yeast being the most frequently isolated. Bacteria and moulds were found in low numbers, whereas yeast were found in numbers up to 10⁷CFU/g. The most frequently isolated yeast, bacteria and moulds belonged to the species of Zygosaccharomyces rouxii, Bacillus subtilis and Aspergillus terreus, respectively.Fifteen isolates were screened for their ability to grow in presence of low a_w (0.65-0.90), low pH (pH=2.0-7.0), ethanol (0-15%), sorbic acid (0-1500ppm) and different temperatures (15°C-25°C) relevant for chocolate manufacturing. Z. rouxii was overall the most tolerant organism to the stress factors and grew within the same range of environmental conditions as found in chocolate pralines. It was able to grow at water activities down to 0.70, ethanol concentrations up to 6.0%, pH down to pH2.0, sorbic acid concentrations up to 1500ppm and at all temperatures tested. Eurotium amstelodami also showed high tolerance towards all the stress factors except for ethanol. None of the bacteria were able to grow at the conditions tested. However, B. subtilis survived the 60day incubation period.