In recent decades, nucleic acid sequencing technologies used for metagenomic analysis have become the main methods for assessing the composition of microbiota. At the same time, the use of novel methods of cultivation and identification of microorganisms in microbiological research led to the renaissance of culture-based technologies, because facilitated the discovery and isolation of both new strains of well-known microorganisms as well as uncultivated and unexplored bacterial taxa. The aim of this study was to evaluate the potential of using the culture-based method for the assessment of the qualitative and quantitative composition of the intestinal microbiota in healthy children. Eleven growth media were inoculated with serial dilutions of stool samples in order to analyze the profile of dominant anaerobic bacteria, as well as aerobic bacteria and fungi in 20 healthy children aged 2–4 years. The identification of microorganisms was performed using MALDI TOF MS and 16S rRNA gene fragment sequencing were used. 1,819 isolated and identified strains belong to 7 phyla, 13 classes, 18 orders, 33 families, 77 genera and 149 species in the Bacteria domain. The Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria phyla were most abundant and frequent. The greatest species diversity (more than 85 species) was found in the Firmicutes phylum. Ten new previously uncharacterized bacterial strains were isolated.