Riverine cyanobacterial blooms are increasing worldwide and are driven in large part by eutrophication. Despite substantial data on nutrient/bloom relationships in lakes and reservoirs, our understanding of nutrient mechanisms driving cyanobacterial blooms in rivers remains limited as rivers can have more complex temporal and spatial nutrient delivery. This study investigated how nutrient conditions influence cyanobacterial dominance and microcystin production in river phytoplankton. Water from the Cumberland River, Tennessee, USA was incubated across four nitrogen (N) and phosphorus (P) gradient scenarios for 36 days to assess how concentration and nutrient ratios influence bloom development. Total algal biomass was co-limited by N and P. Cyanobacteria became dominant with added P (∼1 mg/L) and low N/P molar ratios (<
10.7), but different genera proliferated relative to N availability. N-fixing Dolichospermum sp. dominated at ambient nutrients and very low N/P ratios (0.9) while non-N-fixing Microcystis sp. dominated when both N and P were added. Green algae-dominated communities at the highest N additions and N/P ratios (>
31.8). Algal cell abundance spiked at day 31 in all treatments after much of the initial added nutrients were incorporated into biomass, and corresponded with elevated ammonium in the water. Late-stage (day 31) algal abundance and microcystin in the water were better predicted by initial dissolved nutrient conditions, than concentrations measured at the time of sampling. These results highlight the importance of nutrient co-limitation and N/P ratios on phytoplankton composition and the role of P in promoting Microcystis dominance. N limitation can be a trigger for N-fixing cyanobacteria dominance, and lower toxin synthesis. This study advances our understanding of how nutrient thresholds, ratios, and the timing of nutrient delivery influence cyanobacterial dominance and microcystin production in rivers, which have important implications for the prediction and management of lotic harmful cyanobacterial blooms.