Background: The present work aims to investigate the antioxidant and antimicrobial activities as well as the potential of DCS1 lipopeptides produced by Bacillus methylotrophicus DCS1 strain at inhibition and disruption of biofilm formation. Results: The produced biosurfactants were characterized as lipopeptides molecules by using thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR). The DCS1 lipopeptides were assayed for their antioxidant activity through five different tests. The scavenging effect on DPPH radicals at a concentration of 1 mg mL−1 was 80.6%. The reducing power reached a maximum value of 3.0 (OD700 nm) at 2 mg mL−1 . Moreover, the DCS1 lipopeptides exhibited a strong inhibition of β-carotene bleaching by linoleic acid assay with 80.8% at 1 mg mL−1 and showed good chelating ability and lipid peroxidation inhibition. The in vitro antimicrobial activity of DCS1 lipopeptides showed that they display significant antibacterial and antifungal activities. The anti-adhesive activity of DCS1 lipopeptides was evaluated against several pathogenic microorganisms. The lipopeptides showed excellent anti-adhesive activity, even at low concentrations, in a polystyrene surface pre-treatment against all the microorganisms tested. Further, they can disrupt performed biofilms. Conclusion: This study shows the potentiality of DCS1 lipopeptides as natural antioxidants, antimicrobial and/or anti-adhesive agent for several biomedical and industrial applications.