Healthcare-associated diseases, especially in vulnerable neonates, may be complicated by environmental contamination in neonatal examination room (NER). The bacterial contamination profile and antibiotic susceptibility patterns of isolates obtained from a neonatal examination room were evaluated in this investigation. On May 13, 2025, a cross-sectional study was carried out in the neonatal unit of Al-Bayda Medical Center in Libya. Samples were collected from selected high-touch surfaces, medical equipment, and room air. The passive settle-plate method was used to sample room air, and sterile cotton swabs soaked with sterile normal saline were used to obtain surface samples. Standard microbiological techniques, such as colony morphology, Gram staining, selective media, and biochemical testing, were used to identify the bacterial isolates. The Kirby-Bauer disk diffusion method was used to test for antimicrobial susceptibility in accordance with CLSI M100 (2024) guidelines. 97% (n = 58) of the bacteria were Gram-positive, while only 3% (n = 2) were Gram-negative. Micrococcus species (31.7%), Kocuria species (30.0%), and Bacillus species (26.7%) were the most common isolates. Additionally, clinically significant bacteria were found, such as Pseudomonas spp. (3.3%), Enterococcus spp. (3.3%), and Staphylococcus spp. (5.0%). Among tested isolates, the highest sensitivity percentages were toward gentamicin (98.3%), clarithromycin (95.0%), imipenem (95.0%), ciprofloxacin (93.3%), and ceftriaxone (93.3%). Highest resistant' percentages were shown against ceftazidime (91.7%), oxacillin (68.3%), and erythromycin (58.3%). There was significant bacterial contamination in the neonatal examination room, with Gram-positive organisms predominating. cleaning, disinfection, and infection prevention and control procedures as well as routine environmental microbiological surveillance must be reinforced. ............... Keywords:................ Environmental bacterial contamination, Neonates ICU, Antibiotic sensitivity.