Stenotrophomonas maltophilia is an emerging nosocomial pathogen responsible for several infections in immunocompromised patients. To characterize the antimicrobial resistance and virulence potential of this microorganism in a Brazilian hospital, a total of 936 samples were collected from a nosocomial environment and medical devices, and 100 isolates from clinical specimens were obtained in the same hospital. S. maltophilia was found in 3% of the samples collected, especially in bed rails from hospital rooms. The smf-1 gene was detected in 23% and 42% of the clinical and hospital environment isolates, respectively, and almost all (96.8%) isolates that harbored smf-1 were able to form biofilm. All isolates were susceptible to minocycline and chloramphenicol, and the majority of isolates were susceptible to levofloxacin. High resistance to ceftazidime was detected in both groups of isolates. Resistance to trimethoprim-sulfamethoxazole (TMP/SMX) was found in 14.8% of the isolates. All TMP/SMX-resistant isolates presented class 1 integron and sul1 gene, and 47.4% of them also harbored the sul2 gene, which was inserted into a 7.3 kb plasmid. Genetic relatedness among the isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR, and eight genetic patterns were identified. One pattern comprised 54.7% of isolates and was spread among clinical and environmental (furniture and medical devices) sources. The presence of S. maltophilia in the hospital environment indicates that it can act as a reservoir of this microorganism. In addition, hospital isolates resistant to TMP/SMX showed that the genetic determinants were present in mobile elements, which can constitute great concern, as it may indicate a tendency to spread.