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The present study investigated selected benthic dinoflagellates isolated from different regions from the Western Atlantic, with respect to their morphology, growth, toxicity, and toxin production in culture. A total of nine strains of benthic dinoflagellates belonging to three genera were cultivated: Amphidinium massartii, Amphidinium operculatum (2 strains), Coolia malayensis (2 strains), Prorocentrum hoffmannianum (2 strains), and Prorocentrum mexicanum, whose morphological and genetic characterizations matched previous descriptions, and Prorocentrum sp., morphologically related to P. cf. norrissianum. The two strains of C. malayensis from Brazil attained the highest growth rates (0.42–0.47 day−1), but the lowest cell densities (2.2–2.9 × 104 cells mL−1) in culture. The highest cell densities were recorded for A. massartii from Cuba (3.8 × 105 cells mL−1). All species/strains investigated exhibited moderate toxicity to larvae of the brine shrimp Artemia salina; A. massartii being the most toxic species and Prorocentrum sp. the least one. Additionally, extracts of Prorocentrum species (P. hoffmannianum and Prorocentrum sp.) tested positive in mouse bioassays following intraperitoneal injection. Moderate to high concentrations of okadaic acid (OA), but no dinophysistoxins (DTXs), were found in both P. hoffmannianum strains from Cuba; but no diarrheic toxins were detected in either P. mexicanum from Cuba or Prorocentrum sp. from southern Brazil. Finally, five novel amphidinols were detected in cultures of both A. massartii (Cuba) and A. operculatum (Brazil) by LC-MS/MS, with molecular weights of 1440.8 (two isomers), 1360.8, 1287.7, and 984.6. These findings clearly indicate the need to include benthic species among the harmful microalgae surveyed in regional monitoring programs of phytoplankton.