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Antibiotics are a group of compounds that can inhibit bacterial growth (bacteriostatic) or cause of death bacterial (bactericidal). Bacterial resistance occurs due to inappropriate administration of antibiotic doses and mis of diagnosis by health workers. Bacteria that are resistant to penicillin, cephalosforin and aztreonam generally have an enzyme released to defend themselves, this enzyme is called Extended Spectrum Beta Lactamase (ESBL). This study aims to determine the number of gram-negative bacteria that produce Extended Spectrum Beta Lactamase (ESBL) which is examined from samples that are cultured in the microbiology section in the laboratory of RSUD Prof.DR.W.Z. Johannes Kupang from 2016 to 2018. Secondary data were taken, processed and presented in descriptive form. The Bacteria Extended Spectrum Beta Lactamase (ESBL) was the most found is 104 (50.9%) Klebsiella pneumoniae bacteria, followed by 56 (29.78%) Escherichia coli bacteria, and Enterobacteriaceae (3.61%). ESBL group bacteria mostly came from the Neonatal Intensive Care Unit (NICU) room as many as 26, ESBL group bacteria were mostly found in pus samples as many as 33 (19.41%).