Background/Objectives: Contaminated surfaces play an important role in the nosocomial infection of patients in intensive care units (ICUs). This study, conducted in two ICUs at Edouard Herriot Hospital (Lyon, France), aimed to describe rooms' microbial ecology and explore the potential link between environmental contamination and patients' colonization and/or infection. Methods: Environmental samples were realized once monthly from January 2020 to December 2021 on surfaces close to the patient (bedrails, bedside table, and dedicated stethoscope) and healthcare workers' high-touch surfaces, which were distant from the patient (computer, worktop/nurse cart, washbasin, and hydro-alcoholic solution/soap dispenser). Environmental bacteria were compared to the cultures of the patients hospitalized in the sampled room over a period of ± 10 days from the environmental sampling. Results: Overall, 137 samples were collected: 90.7% of the samples close to patients, and 87.9% of the distant ones were positives. Overall, 223 bacteria were isolated, mainly: Enterococcus faecalis (15.7%), Pantoea agglomerans (8.1%), Enterobacter cloacae/asburiae (6.3%), Bacillus cereus and other Bacillus spp (6.3%), Enterococcusfaecium (5.8%), Stenotrophomonas maltophilia (5.4%), and Acinetobacter baumannii (4.9%). Throughout the study, 142 patients were included, of which, n = 67 (47.2%) were infected or colonized by at least one bacterium. In fourteen cases, the same bacterial species were found both in environment and patient samples, with the suspicion of a cross-contamination between the patient-environment (n = 10) and environment-patient (n = 4). Conclusions: In this work, we found a high level of bacterial contamination on ICU rooms' surfaces and described several cases of potential cross-contamination between environment and patients in real-world conditions.