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Objectives Hospital environments are potential reservoirs of bacteria associated with nosocomial infections. In this study, the distribution of cultivable environmental bacteria of clinical importance from a Malaysian tertiary hospital was determined and their resistotypes and genotypes were investigated. Methods Swab and fluid samples (n = 358) from healthcare workers’ hands, frequently touched surfaces, medical equipment, patients’ immediate surroundings, ward sinks and toilets, and solutions or fluids of 12 selected wards were collected. Biochemical tests, PCR and 16S rRNA sequencing were used for identification following isolation from CHROMagar™ Orientation medium. Clinically important bacteria such as Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter spp., Pseudomonas aeruginosa and Enterobacter spp. were further characterised by disc diffusion method and rep-PCR. Results The 24 Gram-negative and 19 Gram-positive bacteria species identified were widely distributed in the hospital environment. Staphylococci were predominant, followed by Bacillus spp. and P. aeruginosa. Frequently touched surfaces, medical equipment, and ward sinks and toilets were the top three sources of bacterial species. Nine S. aureus, four Acinetobacter spp., one K. pneumoniae and one Enterobacter spp. were multidrug-resistant (MDR). The ESKAPE organisms were genetically diverse and widely dispersed across the hospital wards. A MDR MRSA clone was detected in a surgical ward isolation room. Conclusion The large variety of cultivable, clinically important bacteria, especially the genetically related MDR S. aureus, K. pneumoniae, Acinetobacter spp. and Enterobacter spp., from various sampling sites indicated that the surfaces and fomites in the hospital were potential exogenous sources of nosocomial infection in the hospital.