A novel tetranuclear Dy(III)-based cluster, namely [Dy4(CO3)L4 (acac)2(CH3OH)2(CH2OH)2]·2CH3OH (1) (where H2L is 2-(hydroxyimino)-2-[(5-bromo-2-hydroxyphenyl)methylene]hydrazide and Hacacis acetylacetonate) has been constructed by employing a newly prepared Schiff based ligand H2L. X-ray diffraction data reveal that 1 is mainly consist of a rhombic Dy4core, in which the four Dy3+ ions are bridged by six μ-O atoms from four ligands H2L and two μ2-O atoms from thein situformed CO32−via spontaneous fixation of atmospheric CO2.The crystalstructure of 1 possesses two distinct Dy3+ ions coordinationcenter, and the nine-coordinated Dy73+ionscenter presenting a distorted spherical capped square antiprism geometry and eight-coordinated Dy43+ionscenter presenting a distorted triangular dodecahedron geometry.Magnetic study indicates that 1displays slow magnetic relaxation behavior under Hdc = 0 Oe filed. Furthermore, the fluorescence property investigation reveals that 1shows DyIIIcharacteristic emission peaks. Moreover, theantibacterial activity and mechanism of 1 have been systemically studied, and cluster 1 possesses well antibacterial activity on five common bacteria (Escherichia coli, Staphylococcus aureus, Staphylococcus albus, Bacillus subtilis, and Micrococcus luteus). Inaddition,the interaction between 1 and DNA have been studied.