Stenotrophomonas maltophilia is an opportunistic multidrug resistant pathogen causing hospital-acquired infections (HAIs) with limited treatment options. We aimed to determine the prevalence of S. maltophila causing HAIs and environmental contamination in the intensive care units (ICUs) and wards of Assiut University Hospitals. We determined the antibiotic resistance profiles of, production of metallo-β-lactamases (MBLs) by, and the presence of the sul II gene in these isolates. The study included 362 patients with HAIs and 4151 environmental samples from the ICUs and wards. Antibiotic sensitivities were tested by the disc diffusion method; imipenem minimum inhibitory concentration (MIC) was determined using the E-test. Metallo-β-lactamase enzymes (MBLs) were detected phenotypically by combined disc test (CDT) and double disc synergy test (DDST). The sul ΙΙ gene was detected by polymerase chain reaction. The percentages of S. maltophilia causing infections and environmental contamination were found to be 9.7% and 0.67% respectively. Respiratory tract infection was the most common infection (17.97%). Isolates were highly resistant to aztreonam, penicillins, carbapenems, quinolones, cephalosporins, aminoglycosides, chloramphenicol and tetracyclines, and least resistant to trimethoprim- sulfamethoxazole (SXT). All imipenem resistant isolates (82.54%) showed MBL phenotypically by both tests. For imipenem sensitive isolates (17.46%), MBL was detected by DDST and CDT in 36.36% and 18.18% respectively. Isolates resistant to SXT had sul II genes. In conclusion, S. maltophilia is a significant hospital pathogen at Assiut University Hospitals with high percentages of resistance to many antimicrobials, making the possibility of dissemination worrisome. In our setting, SXT is the agent of choice for the treatment of S. maltophilia infections