© 2020 Elsevier B.V. Harmful algae blooms (HABs) are characterized for the coexistence of phytoplankton species with dynamic and complex biotic interactions (e.g., competition, symbiosis, predation, parasitism, allelopathy), that occur at fine temporal and spatial scales, and are relevant to understand the role that different species of phytoplankton play in the regulation of HABs. In this work the allelopathic effects of Margalefidinium polykrikoides (=Cochlodinium polykrikoides) and Gymnodinium impudicum on Gymnodinium catenatum were evaluated. The allelopathic abilities of M. polykrikoides and G. impudicum were investigated in bi-algal culture experiments and in trials in which target species were co-cultured, separated by a 10 μm membrane to prevent a direct cell-to-cell contact; and also by the addition of different volumes of culture media without cells. For all trials, cells of each species were harvested during exponential phase and cultured together by triplicate at three relative abundances: 1:1 (200 Cells mL–1 of each species, G. catenatum and M. polykrikoides or G. impudicum), 2:1 (400 Cells mL–1 of G. catenatum and 200 Cells mL–1 of M. polykrikoides or G. impudicum), and 1:2 (200 cells mL–1 of G. catenatum and 400 Cells mL–1 of M. polykrikoides or G. impudicum). All bioassays were carried out by triplicate in 250 mL Erlenmeyer flasks with 150 mL of modified GSe medium with an initial inoculum of 200 or 400 Cells mL−1. During experiments G. catenatum abundances were enumerated daily. In bi-algal culture experiments mortalities of G. catenatum were from 50% to 100% after 48 h of cell contact with M. polykrikoides or G. impudicum. In the case of culture media without cells, only M. polykrikoides caused a decrease in the cell abundance and growth rate of G. catenatum. Morphological changes occurred in G. catenatum when in contact with M. polykrikoides and G. impudicum, such as membrane shedding, prominent nucleus, loss of flagella, cell lysis, as well as the separation of long chains into individual cells. These results suggest that in the natural environment M. polykrikoides and G. impudicum have allelopathic interactions in G. catenatum, which could negatively affect its growth and survival, indicating that these species could displace blooms of G. catenatum.