| Summary: | Brazilian fish farming goes together with the emergence of numerous bacterial diseases, with <i>Aeromonas hydrophila</i> being the main bacterial pathogen. As a consequence, antimicrobials are excessively used. Considering that antimicrobials are relatively stable and nonbiodegradable, medicinal plants and their phytochemicals have been used as alternative treatments of bacteriosis in fish farming. Limonene is a monoterpene available in two enantiomers: R-(+)-limonene and S-(-)-limonene. This study analyzed the antibacterial activity of the phytochemicals S-(-)-limonene and R-(+)-limonene against some bacteria isolated from silver catfish (<i>Rhamdia quelen</i>). Furthermore, by means of spectrophotometry and atomic force microscopy, we also investigated the combination therapy of phytochemicals with antimicrobials and their activity in terms of inhibiting biofilm formation. Six clinical isolates and a standard strain were selected for antimicrobial activity testing. Biofilm formation was tested in 96-well plates and nylon cubes. The most sensitive of the strains tested was the <i>A. hydrophila</i> strain (MF 372510). S-(-)-limonene and R-(+)-limonene had high minimum inhibitory concentrations; however, they strongly inhibited <i>A</i>. <i>hydrophila</i> biofilm formation. R-(+)-limonene and S-(-)-limonene had an additive effect when combined with florfenicol and an antagonistic effect with oxytetracycline. In general, the phytochemicals tested showed strong antibiofilm activity against <i>A</i>. <i>hydrophila</i>, and when in combination therapy with florfenicol, they showed an additive effect against the treatment of <i>A</i>. <i>hydrophila</i>.
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