Neonatal intensive care units (NICUs) are specialized health care settings where neonates with critical conditions and preterm births are exposed to a high risk of healthcare-associated infections (HAI). Non-critical medical equipment and frequently touched surfaces in the NICU environment may act as a medium of bacterial transmission. It is important to know the level of bacterial contamination in the NICU environment in order to enhance infection prevention and control. The objectives of the present study were to determine the level of bacterial contamination in the NICU environment in Iraq, to determine the distribution of Gram-positive and Gram-negative bacteria and to determine the most common bacteria present in the NICU environment. A cross-sectional survey was carried out in the NICU of Al-Rifai General Hospital in Al-Rifai City in Iraq from November 2023 to March 2024. A total of 61 swab samples were collected from frequently touched surfaces and frequently used non-critical equipment in the NICU environment. These surfaces and equipment include incubators, suction tips, stethoscopes, door handles, medical equipment, and hands of healthcare workers. These samples were then cultured using standard microbiological practices and incubated at 37°C for up to 48 hours. The bacteria were identified based on their colonial characteristics and Gram staining. Among the total samples, 58 (95.1%) had evidence of bacterial growth, while 3 (4.9%) had no growth. Of the culture-positive samples, Gram-positive organisms were predominant, accounting for 40 (68.9%), while Gram-negative organisms accounted for 18 (31.0%). Among Gram-positive organisms, the most prevalent organism was Staphylococcus aureus, accounting for 13 (32.5%), followed by Bacillus cereus (6, 15.0%), Staphylococcus epidermidis (4, 10.0%), and Enterococcus faecalis (4, 10.0%). Among Gram-negative organisms, Escherichia coli was the predominant organism, accounting for 10 (55.5%), followed by Acinetobacter baumannii (3, 16.6%), and Pseudomonas aeruginosa (2, 11.1%). From the qualitative mapping, it was observed that predominant organisms were isolated from high-touch surfaces and non-critical equipment, including incubators, suction tips, stethoscopes, door handles, and healthcare workers’ hands. The study revealed a high level of environmental contamination in the NICU, with a predominance of Gram-positive organisms and clinically important Gram-positive and Gram-negative organisms. The study also revealed that predominant organisms are associated with high-touch surfaces and non-critical equipment, emphasizing the need for strict hand hygiene and environmental decontamination practices in the NICU setting.