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Bismuth oxide is an important bismuth compound having applications in electronics, photo-catalysis and medicine. At the nanoscale, bismuth oxide experiences a variety of new physico-chemical properties because of its increased surface to volume ratio leading to potentially new applications. In this manuscript, we report for the very first time the synthesis of bismuth oxide (Bi 2 O 3 ) nano-flakes by pulsed laser ablation in liquids without any external assistance (no acoustic, electric field, or magnetic field). The synthesis was performed by irradiating, pure bismuth needles immerged in de-ionized water, at very high fluence ∼160 J cm −2 in order to be highly selective and only promote the growth of two-dimensional structures. The x - and y -dimensions of the flakes were around 1 μm in size while their thickness was 47.0 ± 12.7 nm as confirmed by AFM analysis. The flakes were confirmed to be α- and γ-Bi 2 O 3 by SAED and Raman spectroscopy. By using this mixture of flakes, we demonstrated that the nanostructures can be used as antimicrobial agents, achieving a complete inhibition of Gram positive (MSRA) and Gram negative bacteria (MDR-EC) at low concentration, ∼50 ppm.