Microbial contamination of hospital environment plays an important role in the spread of health care-associated infections (HCAIs). This study was conducted to determine bacterial contamination, bacterial profiles and antimicrobial susceptibility pattern of bacterial isolates from environmental surfaces and medical equipment. A cross-sectional study was conducted at Tikur Anbessa Specialized Hospital (TASH) from June to September, 2018. A total of 164 inanimate surfaces located at intensive care units (ICUs) and operation theaters (OTs) were swabbed. All isolates were identified by using routine bacterial culture, Gram staining and a panel of biochemical tests. For each identified bacteria, antibiogram profiles were determined by the Kirby Bauer disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Out of the 164 swabbed samples, 141 (86%) were positive for bacterial growth. The predominant bacteria identified from OTs and ICUs were S. aureus (23% vs 11.5%), Acinetobacter spp (3.8% vs 17.5%) and Coagulase negative Staphylococcus (CONS) (12.6% vs 2.7%) respectively. Linens were the most contaminated materials among items studied at the hospital (14.8%). The proportions of resistance among Gram-positive bacteria (GPB) were high for penicillin (92.8%), cefoxitin (83.5%) and erythromycin (54.1%). However, the most effective antibiotics were clindamycin with only 10.4% and 16.5% resistance rates, respectively. The antimicrobial susceptibility profiles of Gram-negative bacteria (GNB) revealed that the most effective antibiotics were amikacin, ciprofloxacin, and gentamicin with resistance rate of 25%, 37.5%, and 46.3%, respectively. However, the highest resistance was recorded against ampicillin (97.5%), ceftazidime (91.3%), ceftriaxone (91.3%) and aztreonam (90%). The inanimate surfaces near immediate patient environment and commonly touched medical equipment within OTs and ICUs are reservoirs of potential pathogenic bacteria that could predispose critically ill patients to acquire HCAIs. The proportions of antimicrobial resistance profile of the isolates are much higher from studied clean inanimate environments.