Due to their nutritional quality, microalgae play a crucial role in aquaculture, being used in the feeding of bivalve mollusks, zooplankton, and in the larval stages of crustaceans and fish. However, harvesting microalgae by centrifugation is an energy-intensive process. Flocculation, on the other hand, is a low-cost method that allows for high cell recovery, often exceeding 90%. This study aimed to analyze the flocculation efficiency of the microalga Tetraselmis chuii, using alkaline flocculation (sodium hydroxide) and chemical flocculation with natural organic polymers (Tanfloc SG, Tanfloc MT, Tanfloc MTH, and chitosan) and synthetic polymers (Flopam), based on efficiency, floc size, temperature effect, sedimentation, and cost. The microalga was cultured in f/2 medium and monitored by measuring optical density. Flocculation was carried out using jar tests. Initially, a screening was performed with all flocculants at concentrations of 70, 150, and 200 mg/L, except for chitosan (3,000 to 7,000 µl/L). The flocculants MTH, SG, and sodium hydroxide were selected for the optimization stage, with concentrations tested at 5, 70, 135, 200, 265, 330, 395, and 460 mg/L. The effect of temperature (5°C and 30°C) was later assessed to simulate outdoor cultivation in winter and summer. Sedimentation rate, concentration factor, and aggregate volume index were calculated. Cost analysis was based on concentration, temperature, and efficiency. ImageJ software was used to measure floc size using the Feret diameter. The results showed that the flocculant Tanfloc SG at 22°C exhibited the highest flocculation efficiency (>90%) at a concentration of 330 mg/L, the highest concentration factor (24.100 ± 0.41), the lowest cost ($1.63), and a good sedimentation rate (0.088 cm/s). MTH showed higher efficiency at higher temperatures (30°C), along with a greater sedimentation rate (0.121 cm/s) and a good concentration factor (13.393 ± 1.263). In contrast, sodium hydroxide was the most suitable flocculant under low-temperature conditions; however, it produced smaller flocs (31–50 µm), had the lowest concentration factor and sedimentation rate, and was the most expensive flocculant.