Abstract: Off-flavor contamination constitutes a persistent constraint on tilapia aquaculture development, with microorganisms widely regarded as the principal producers of off-flavor metabolites. To elucidate the coupling between pond sediment microbial communities and the accumulation of the two principal off-flavor compounds, geosmin (GSM) and 2-methylisoborneol (2-MIB), in Nile tilapia (Oreochromis niloticus) aquaculture ponds, we collected the water, fish muscle, and sediment samples monthly from two farms in Maoming, Guangdong in June, July, and September, 2025. Then, we measured GSM and 2-MIB concentrations, characterized sediment microbial community structure by metagenomics, annotated the terpenoid methylerythritol 4-phosphate (MEP) biosynthesis pathway and key off-flavor biosynthesis genes, and quantified the correlations between microbial community composition and off-flavor compound concentrations. GSM and 2-MIB concentrations in water and fish muscle exhibited significant farm-by-time interaction effects and increased progressively over the aquaculture cycle. Pseudomonadota was the dominant phylum in the sediment microbial community. Actinomycetota, the principal putative odor-producing phylum, comprised approximately 7% of the community. Cyanobacteriota was detected at low relative abundance. Alpha diversity of sediment microbial communities did not differ significantly between farms. Community composition was significantly shaped by farm identity and sampling month (p<0.01), with the discriminant taxa consisting predominantly of anaerobic, nutrient-cycling, non-odor-producing lineages. Although a complete MEP precursor biosynthesis pathway was detected across all sediment samples, the terminal off-flavor synthase genes (geoA, gppmt, mibs) were universally absent. Neither Actinomycetota relative abundance nor temporal community dynamics corresponded to off-flavor concentrations in water or fish muscle, further corroborating the functional gene evidence. In summary, these findings indicate that sediment microorganisms in typical Nile tilapia ponds in Maoming harbor have no genetic capacity for GSM and 2-MIB biosynthesis and do not function as an endogenous off-flavor reservoir. Planktonic microorganisms may contribute to off-flavor formation, which requires further validation. These results define the functional scope of pond sediment in off-flavor formation, providing a theoretical framework for precision off-flavor source attribution, prioritization of water column algal control, and rationalization of sediment management practices.