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Background: Microbial resource orchid is a collection of Gram-positive and Gram-negative clinical isolates sourced from different hospitals and diagnostic laboratories in India. We determined the antibiotic susceptibility of a set of Gram-negative Enterobacteriaceae and non-fermenter clinical isolates from microbial resource orchid, collected during the period of 2002-2012 against commonly used antibiotics., Materials and Methods: A total of 247 Gram negative strains consisting of 142 Enterobacteriaceae and 105 non-fermenters from microbial resource orchid were selected for determining minimum inhibitory concentration against β-lactams, aminoglycosides, quinolone, and tetracycline by agar dilution method as per clinical and laboratory standards institute guidelines., Results: All the isolates had high resistance to ampicillin, piperacillin, ceftazidime, gentamicin, tetracycline, and ciprofloxacin. Pseudomonas aeruginosa showed moderate resistance to carbapenems., Conclusion: This study demonstrated the high level of antibiotic resistance among the strains collected under microbial resource orchid and further, such data and the strains can be used in new chemical entities profiling.