Microbial communities in freshwater lake sediments exhibit a distinct depth-dependent variability. Further exploration is required to understand their biodiversity pattern and microbial interactions in vertical sediments. In this study, sediment cores from two freshwater lakes, Mugecuo (MGC) and Cuopu (CP), on the Tibetan plateau were sampled and subsequently sliced into layers at a depth of every centimeter or half a centimeter. Amplicon sequencing was used to analyze the composition, diversity, and interaction of microbial communities. Results showed that sediment samples of both lakes could be clustered into two groups at a sediment depth of about 20 cm, with obvious shifts in microbial community compositions. In lake MGC, the richness component dominated β-diversity and increased with depth, indicating that the microbial communities in the deep layer of MGC was selected from the surface layer. Conversely, the replacement component dominated β-diversity in CP, implying a high turnover rate in the surface layer and inactive seed banks with a high variety in the deep layer. A co-occurrence network analysis showed that negative microbial interactions were prevalent in the surface layers with high nutrient concentrations, while positive microbial interactions were more common in the deep layers with low nutrient concentrations, suggesting that microbial interactions are influenced by nutrient conditions in the vertical sediments. Additionally, the results highlight the significant contributions of abundant and rare taxa to microbial interactions and vertical fluctuations of β-diversity, respectively. Overall, this work deepens our understanding of patterns of microbial interactions and vertical fluctuation in β-diversity in lake sediment columns, particularly in freshwater lake sediments from the Tibetan plateau.