The Tabuk region is located in the northern part of Saudi Arabia, and it has an area of 117,000 km2 between longitudes 26° N and 29° N and latitudes 34° E and 38° E. King Salman Bin Abdulaziz Royal Natural Reserve (KSRNR) is the largest natural reserve in Saudi Arabia and covers about 130,700 km2. It represents a new tourist attraction area in the Tabuk region. Human activities around the lake may lead to changes in water quality, with subsequent changes in microenvironment components, including microbial diversity. The current study was designed to assess possible changes in bacterial communities of the water sediment at some natural lakes and artificial waterpoints of KSRNR. Water samples were collected from ten different locations within KSRNR: W1, W2, W3 (at the border of the royal reserve); W4, W5, W6, W7 (at the middle); and W8, W9, and W10 (artificial waterpoints). The total DNA of the samples was extracted and subjected to 16S rRNA sequencing and metagenomic analysis; also, the environmental parameters (temperature and humidity) were recorded for all locations. Metagenomic sequencing yielded a total of 24,696 operational taxonomic units (OTUs), which were subsequently annotated to 193 phyla, 215 classes, 445 orders, 947 families, and 3960 genera. At the phylum level, Pseudomonadota dominated the microbial communities across all samples. At the class level, Gammaproteobacteria, Clostridia, Alphaproteobacteria, Bacilli, and Betaproteobacteria were the most prevalent. The dominant families included Enterobacteriaceae, Pseudomonadaceae, Clostridiaceae, Comamonadaceae, and Moraxellaceae. At the genus level, Pseudomonas, Clostridium, Acinetobacter, Paenibacillus, and Acidovorax exhibited the highest relative abundances. The most abundant species were Hungatella xylanolytica, Pseudescherichia vulneris, Pseudorhizobium tarimense, Paenibacillus sp. Yn15, and Enterobacter sp. Sa187. The observed species richness revealed substantial heterogeneity across samples using species richness estimators, Chao1 and ACE, indicating particularly high diversity in samples W3, W5, and W6. Current study results help in recognizing the structure of bacterial communities at the Tubaiq area in relation to their surroundings for planning for environmental protection and future restoration of affected ecosystems. The findings highlight the dominance of various bacterial phyla, classes, families, and genera, with remarkable species richness in some areas. These results underscore the influence of human activities on microbial diversity, as well as the significance of monitoring and conserving the reserve’s natural ecosystems.