The thawing of frozen soils has resulted in the release of tremendous amounts of organic carbon. Permafrost-derived dissolved organic carbon (DOC) has been demonstrated to be biolabile to microbes in aquatic ecosystems. Cladoceran grazing is another regulatory force that may affect the properties and/or characteristics of the bacterial community. However, the effects of permafrost DOC and cladoceran grazing on bacterial abundance, community composition, and diversity in aquatic ecosystems have rarely been explored. In this study, DOC was extracted from frozen soils adjacent to the shoreline of a pristine alpine lake and used to culture bacteria. The bacterial abundance decreased over time during the bioincubation experiment, whereas Daphnia feeding had no significant effect on the bacterial abundance. The genera Pedobacter, Novosphingobium, Flavobacterium and Limnohabitans had relatively high abundances after a 27-d bioincubation period. The overall decrease in bacterial diversity in the Daphnia grazing experiment was higher than that in the bacterial incubation experiment. The co-occurrence network demonstrated that terrestrial and microbial humic-like components C1 and C2 had strong interactions with Pseudolabrys, and tyrosine-like component C3 presented correlations with Hyphomicrobium and Vampirovibrio. Bacterial communities in the two experiments were significantly different. Sphingomonadales, Sphingobacteriales, Burkholderiales, and Actinomycetales were the primary contributors to these differences. Our study contributes significantly to a broader understanding of carbon cycling and microbial food web in lakes, particularly in high-altitude and polar lakes.