BackgroundMicrobial contamination of hospital environment, especially in operating theatres (OT) and other specialized units has greatly contributed to continuous and multiple exposure to nosocomial infections by patients and the public.We purposed to assess microbial contamination of operating theatres and antibacterial sensitivity pattern of bacteria isolated from theatres of Mbale Regional Referral Hospital, Eastern Uganda.MethodsWe employed a laboratory based cross-sectional study design. Swabbing of different surfaces and settle plate establishment in 4 various operating theatres was carried out. A total of 109 samples were collected, 31 air samples and 78 swabs from four operating theatres. Samples were collected in the mornings after disinfection prior to start of daily operations. Antibacterial sensitivity testing of isolated bacterial pathogens was performed by Kirby Bauer disc diffusion method following standard operating procedure. Colony counts for the settle plates were carried out using a colony counter.ResultsAll the four theatres had their mean colony counts exceeding the acceptable limit of 5 cfu/dm2/h. Gynaecology theatre had up to 261 cfu/dm2/h and Ophthalmology operating theatre had approximately 43 cfu/dm2/h. A total of 14 different organisms were isolated with Pseudomonas spp. [23.9%]; Bacillus spp. [17.5%] and Aspergillus spp. [15.8%] being the most common contaminants respectively. Other isolates included Enterococcus spp., Rhizopus spp. and Coagulate Negative Staphylococcus isolates especially from settle plates. Most bacterial isolates showed considerable resistance to antibacterial agents. Pseudomonas spp. was resistant to chloramphenicol (53.6%) and cotrimoxazole (57.1%). Most of the bacterial pathogens were sensitive to imipenem [83.3%].ConclusionsThere is moderate contamination of operating theatres of Mbale Regional Referral Hospital. Common organisms were Pseudomonas, Bacillus, and Aspergillus spps. Resistance was observed against chloramphenicol and cotrimoxazole. More caution is necessary to carefully disinfect the operating theatres at Regional referral settings and similar tertiary health care centres with more emphasis on obstetrics and gynecology theatres. Diagnosis and care of patients at such clinical settings should consider the possibility of antibiotic resistance.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3374-4) contains supplementary material, which is available to authorized users.