Background: Stenotrophomonas maltophilia is an emerging multiresistant nosocomial pathogen. Aim: This study aimed to isolate S. maltophilia and estimate the frequency of biofilm forming as a virulence factor contributing with resistance to some antibiotics. Methods: A total of 350 samples were collected from (100) patients in different hospitals of Mosul city during the period from April 2022 to June 2023. Using conventional methods and Vitek 2 automated system. Results: The highest isolation rate of S. maltophilia was from blood samples at rate of 51.5%, urine and wound 28.5% , 20% respectively while there was no isolates in pharynx samples, the disc diffusion method used to confirm the resistant of bacterial strains against 10 antimicrobial agents, most of them showed high resistant rates to Amikacin (57.1%), Ceftazidime (62.8%), Ciprofloxacin (85.7%), Piperacillin (77.1%), Cefepime and Gentamicin (71.4%), while it was low against Imipenem (42.8), Meropenem (45.7%) and Cefotaxime (45.71%) also for Piperacillin-tazobactam (48.5%), The resistance level was > 60 % for the most tested antimicrobial agents. The biofilm formation was implemented using microtiter plate method, about (31.43%) of the isolates induced strong level of biofilm formation, (42.85%) moderate while only (25.72%) showed weak intensity of biofilm formation, Conclusion:  The high resistance of  Stenotrophomonas maltophilia to most of the antibiotics used is an indicator that requires attention, in addition most of the resistant isolates had strong biofilm production properties which refer to the virulently of this bacteria relating with serious nosocomial infections.