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Biosurfactants (BSs) are surface-active compounds produced by diverse microorganisms, including the genus <i>Bacillus</i>. These bioactive compounds possess biological activities such as antiadhesive, antimicrobial and antibiofilm effects that can lead to important applications in combating many infections. Based on these findings, we decided to investigate the antibiofilm activity of BSs from the marine <i>Bacillus amyloliquefaciens</i> against <i>Staphylococcus aureus</i> CCM 4223. Expression of biofilm-related genes was also evaluated using qRT-PCR. Isolated and partially purified BSs were identified and characterized by molecular tools and by UHPLC-DAD and MALDI-TOF/MS. <i>Bacillus amyloliquefaciens</i> 3/22, that exhibited surfactant activity evaluated by oil spreading assay, was characterized using the 16S rRNA sequencing method. Screening by PCR detected the presence of the <i>sfp</i>, <i>srfAA, fenD</i> and <i>ituD</i> genes, suggesting production of the lipopeptides (LPs) surfactin, fengycin and iturin. The above findings were further supported by the results of UHPLC-DAD and MALDI-TOF/MS. As quantified by the crystal violet method, the LPs significantly (<i>p</i> < 0.001) reduced biofilm formation of <i>S. aureus</i> in a dose-dependent manner and decreased expression of biofilm-related genes <i>fnbA</i>, <i>fnbB</i>, <i>sortaseA</i> and <i>icaADBC</i> operon. Data from our investigation indicate a promising therapeutic application for LPs isolated from <i>B. amyloliquefaciens</i> toward prevention of <i>S. aureus</i> biofilm infections.