Multidrug resistant (MDR) infections have been a world health issue for many decades, and therefore exploration of new antibiotics to overcome this issue is urgently needed. Regarding antimicrobial properties in the coral holobiont, coral-associated bacteria are suggested as potential producer of new antibacterial agents against MDR infections. The purposes of this study include isolation and identification of pigmented coral-associated bacteria, preparation of pigmented extract and evaluation of its antibacterial property, as well as the characterization of the pigmented extract using thin layer chromatography (TLC). Nine bacteria candidates were isolated from an unidentified stony coral collected from Tanjung Gelam, Karimunjawa National Park, Jepara Region, Central Java Province, Indonesia. Bacterial pigment and other metabolites were extracted using 1-butanol, ethyl acetate and acetone. Silica gel-based TLC was applied to detect β-carotene and characterize the bacterial crude extracts. Antibacterial activity was evaluated against the extended spectrum β-lactamase (ESBL) E. coli, Klebsiella pneumoniae, and MRSA (methicillin-resistant Staphylococcus aureus). A yellow pigmented bacterium was isolated and identified as Vibrio owensii TNKJ.CR.24-7. Nonribosomal peptide-synthetase Peptide-Synthetase NRPS genes fragments were detected. All bacterial extracts from 1-butanol, ethyl acetate and acetone contained the yellow pigment that was identified as β-carotene. Only the crude extract from ethyl acetate inhibited all of MDR bacteria. TLC chromatogram showed that there were 7 bands from 1-butanol extract and 6 bands from ethyl acetate extract. However, crude extract from acetone was not well separated using silica-based TLC. Bands at Rf 0.64 and 0.81 were found both in 1-butanol and ethyl acetate extracts. Keywords: Antibacterial, β-Carotene, MDR, NRPS, Vibrio