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Aeromonas is a Gram-negative bacterium that lives in aquatic habitats. It can be infective in humans. One of its remarkable attributes is the ability of biofilm formation. Many factors are involved in the construction of biofilms as has been described for Pseudomonas, Klebsiella, and Vibrio, among others. The aim of this work was to study the bacterial morphology during the establishment of biofilm through scanning electron microscopy (SEM) and transmission electron microscopy (TEM) with a modified microtiter plate assay and to determine the best conditions for the establishment of Aeromonas caviae Sch3 biolfilm in vitro. We observed several phenotypic changes, including surface appearance, size, presence of extracellular vesicles from 100 to 250 nm in diameter, and flagella. The best conditions for biofilm formation were to grow cultures at 28℃ at pH 6, as determined by the crystal violet assay. This is, to the best of our knowledge, the first study that describes the cell’s biological events involved in the establishment of biofilm formation of Aeromonas caviae Sch3 in vitro.