Quantitative phytoplankton data was collected as part of an interdisciplinary and multiagency experimental study investigating harmful algal bloom (HAB) dynamics in the Caloosahatchee River located in southwestern Florida. Algae and cyanobacteria play crucial roles in aquatic ecosystems, but under favorable environmental conditions such as nutrient enrichment certain taxa may experience population growth booms resulting in HABs. HABs are becoming increasing common in the Caloosahatchee and the Lake Okeechobee system, yet the drivers and processes controlling these events is still under investigation. Here, three independent mesocosm experiments were conducted on the Caloosahatchee River at the W. P. Franklin Lock and Dam, Florida, in February 2021, May 2021, and July 2021 to test the effects of nutrient enrichment of phytoplankton assemblage structure at different times in the year to capture natural temporal variability in water quality and phytoplankton assemblages. The experimental installation consisted of three floating metal cradles each containing four fiberglass chambers filled with native river water. The twelve chambers were assigned to one of five treatments: phosphate (P), nitrate (N), urea (U), ammonium (A), or control (C). Only four of the five treatments were selected for a given experiment: February 2021 treatments included P, N, A, and C, May 2021 treatments included P, U, A, and C, and July 2021 treatments included U, N, A, and C. The P, N, U, and A treatment chambers were enriched in the appropriate nutrient by applying a concentrated dosing solution of either dibasic dodecahydrate sodium phosphate, anhydrous sodium nitrate, solid crystal urea, or liquid ammonium hydroxide respectively to elevate concentrations above ambient levels. The mesocosms were treated and sampled approximately every 24 hours over a 72-hour period (Time 0, Time 24, Time 48, Time 72). May and July 2021 mesocosm were also sampled a week from the initial dosing (T240). Water quality, phytoplankton, cyanotoxin, and environmental DNA (eDNA) samples were collected before and after dosing each day. The current dataset contains only the phytoplankton data including absolute and relative abundance measures at the genus to species level as natural units (NU/mL), cell densities (cells/mL), and biovolumes (um3/mL).