Background: Antibiotic resistance Gram-negative bacteria are becoming responsible for the increased morbidity and mortality, particularly in hospitalized patients. With the increasing use of electronic healthcare, shared use of medical equipment and imperfect hand hygiene, the role of frequently touched environmental surfaces for the potential dissemination of these resistant organisms is now of greater importance, posing a real challenge to conventional infection-control practices. We sought therefore to determine the prevalence and antibiotic resistance pattern of Gram-negative pathogens from inanimate hospital sources. Methods: Environment swab samples were collected from Lagos Island maternity hospital for the isolation of Gram-negative pathogens. Antibiotic susceptibility test, multiple antibiotic indexing, phenotypic and PCR genotypic confirmation of ESBL, carbapenemase gene and plasmid profiling were used to detect and determine the resistance pattern of these isolates. Result: A total prevalence of 35(40.2%) Gram-negative pathogens were recorded in this study. Among the isolates, 33(94.3%) were resistant to ceftriaxone and 23(65.7%) resistant to cefepime, 19(54.3%) were resistant to ciprofloxacin and 21(60%) were resistant to ofloxacin. Absolute resistance of 100% was observed among gentamicin, tigecycline and ampicillin. The carbapenems showed the least resistance where 15(42.9%) were resistant to imipenem, 11(31.4%) doripenem and 10(28.6%) to meropenem. A similar trend of a very high multiple antibiotics resistance (MAR) index was observed among the isolates from all sampling areas. Out of the 35 isolates, 20 (57.1%) were identified as ESBL producers while 4 (11.4%) phenotypically emerged as carbapenamase producers. Genotypically 4 (20%) carried the blaCTX-M gene and 1 (5%) carried blaTem gene while out of 6 Pseudomonas aeruginosa, 2(33.3%) carried Integron 1 gene. All isolates showed no carbapenamase gene while 11(55%) showed the presence of plasmid with a high band size of 23130kbp.Conclusion: This study established the detection of the extended-spectrum beta-lactamase (ESBL) gene indicating an increase in the rate of antibiotic resistance in Gram-negative bacteria and their occurrence in inanimate hospital environments.