Sedimentary DNA (sedaDNA), pollen, and charcoal records from two sediment cores along the lower Negro River floodplain revealed complementary ecological and hydrological patterns throughout the Holocene in the main blackwater river located in central Amazonia. The sedaDNA record from the Lake Pacú sediment core (~9440–370 cal yr BP) provides unprecedented insight into microbial and planktonic communities across millennial-scale environmental changes. During the early Holocene (~9440–8852 cal yr BP), the presence of planktonic diatoms (Discostella nipponica, Melosira varians) and ciliates (Rimostrombidium sp., Strombidium sp.) indicate shallow, moderately productive waters with relatively low acidity compared with current Negro River conditions. A transition from ~8852 to 4520 cal yr BP is characterized by increased biological diversity compared to the early Holocene, with higher abundances and taxonomic richness of diatoms, ciliates, rotifers (Brachionus sp., Asplanchna brightwellii), and Chlorophyta (Pyramimonas tetrarhynchus). These assemblages suggest episodes of elevated nutrient input, temporary water column stratification and hydrological connectivity with surrounding floodplain environments. This interval reflects a dynamic limnological regime, with productivity fluctuating under seasonal flooding and broader hydroclimatic variability. The Late Holocene interval (~4520–370 cal yr BP) shows a pronounced ecological shift. Particularly around ~3000 cal yr BP, sedaDNA reveals the occurrence of mesotrophic diatoms, green algae, rotifers and ciliates, taxa not found under the acidic, humic waters of the Negro River. These conditions were likely driven by river connectivity, changes in water level and flow from tributaries such as the Branco River, whose chemical properties differ significantly from the Negro River. After this interval, these taxa decline toward the most recent samples, reflecting a return to more acidic, low productivity conditions similar to the river today. Complementarily, palynological data from the Apuaú River sediment core (~6450–3540 cal yr BP), a left bank tributary of the Negro River, document simultaneous expansion of Várzea type vegetation and the presence of mesotrophic diatoms (~13%), reinforcing a regional pattern of increased nutrient flux and hydrological heterogeneity during the mid- to late Holocene. Additionally, charcoal peaks dated to ~3320–2620 cal yr BP indicate intensified fire activity during the late Holocene, most likely associated with a regional dry phase rather than anthropogenic activity. Overall, our multi-proxy reconstruction of the lower Negro River provides a rare molecular record throughout the Holocene, revealing shifts in aquatic communities, vegetation and fire regimes in central Amazonia.