Rhizosphere microorganisms play central roles in nutrient cycling and contaminant transformation in sediment-associated freshwater systems, yet their responses to newer pesticides remain insufficiently characterized. In this study, a 28-day Vallisneria natans–rhizosphere sediment microcosm was used to compare the effects of trifluenfuronate and fluopyram at nominal concentrations of 0.01, 0.1, and 1 mg L⁻¹. Bacterial community composition was assessed using 16S rRNA gene sequencing, and shotgun metagenomic data were used to evaluate relative functional annotation patterns. Plant physiological traits and rhizosphere sediment enzyme activities were measured as ecological context for interpreting microorganism-associated responses. Fluopyram, particularly at 1 mg L⁻¹, produced clearer ordination-level shifts in rhizosphere bacterial community composition than trifluenfuronate, although pairwise treatment separation was not statistically resolved after multiple-testing correction. Annotation-based metagenomic profiles also differed between the two pesticides: stronger exposure was associated with reduced relative signals for several xenobiotic-, transport-, and regulation-related annotations, while high-dose fluopyram showed a methane-metabolism-related annotation signal and high-dose trifluenfuronate showed relative enrichment of secondary-metabolism-related annotations. These microbial and annotation-profile responses coincided with stronger inhibition of V. natans growth and greater suppression of rhizosphere sediment enzyme activities under fluopyram exposure. Overall, fluopyram induced more consistent microorganism-associated response patterns than trifluenfuronate in the tested rooted macrophyte–sediment microcosm. The results highlight the sensitivity of rhizosphere microbial communities and metagenomic annotation profiles to pesticide exposure in sediment-associated freshwater systems.