Over the last half-century, microbial communities of the Kamchatka hot springs have been largely studied using molecular, radioisotopic, and cultural approaches. Generally, these results were obtained for mixed samples of water with sediments, for only hydrothermal water, or for only sediment samples. Simultaneous comparative analysis of the microbial communities of water and sediments was performed for only one Kamchatka hot spring with circumneutral pH. Here, the microbial communities of both sediments and water (separately) of hot spring #4229 (the Uzon Caldera, Kamchatka) with a temperature of 50–56 °C and pH of 3.2 were analyzed by 16S rRNA gene V4 fragment amplicon sequencing. It was revealed that the microbial community of sediments was represented by uncultured phylogenetically deep-branching lineages of archaea, such as ARK-15 within Thermoplasmatota and ‘Ca. Marsarchaeales’ from the Thermoproteota phyla. Metagenome analysis showed that these archaea most probably carried out the degradation of organic matter. The microbial community of the hot water is represented by thermoacidophilic, (micro)aerobic, chemolithoautotrophic, hydrogen- and sulfur-oxidizing bacteria of the genera Hydrogenobaculum (phylum Aquificota) and Acidithiobacillus (phylum Pseudomonadota). Radioisotopic tracing and DNA-stable-isotope probing techniques proved their role as primary producers in the hot spring. The experiment revealed significant differences in the composition and functions of the microbial communities of sediments and water through the example of a typical acidic hot spring in Kamchatka.