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This study aimed to investigate the antimicrobial, antibiofilm, and cytotoxic effects of biosurfactant lipopeptides synthesized by Bacillus subtilis TR47II. For this purpose, the lipopeptides were partially purified using a three-step process and characterized. In the first step, the crude extract obtained from acid precipitation exhibited strong antibacterial activity against the Gram-negative opportunistic pathogens Alcaligenes faecalis ATCC 8750, Achromobacter xylosoxidans ATCC 13138, Pseudomonas alcaligenes ATCC 14909, and Pseudomonas putida ATCC 15175. Moreover, partial inhibition was observed against Klebsiella aerogenes ATCC 13048 (42%), Escherichia coli ATCC 25922 (16%), and Pseudomonas aeruginosa ATCC 27853 (47%). The lipopeptides in the crude extract were extracted with methanol and fractioned on a silica gel chromatography column, rendering four TLC-pooled chromatographic fractions, named F1, F2, F3, and F4. The chromatographic fraction F4 was the most bioactive, with MIC values between 300 and 600 µg mL−1. Besides, F4 at sub-MIC doses dislodged the biofilms of A. faecalis, A. xylosoxidans, and P. alcaligenes by about 100, 85, and 81%, respectively. No cytotoxic effect was observed in mammalian cells at MIC. MALDI-TOF-MS analysis revealed that F4 contained cyclic lipopeptides belonging to two families: iturins (m/z 1004 to 1087) and fengycins (m/z 1424 to 1545). The dual effect of F4 on planktonic and sessile growth could suggest that the synergistic application of these biosurfactants could be efficient in the control of these opportunistic pathogens. [ABSTRACT FROM AUTHOR]