The present study aimed to explore the genotypic and phenotypic characteristics of biofilm formation in Bulgarian nosocomial Stenotrophomonas maltophilia isolates (n 5 221) during the period 2011-2022, by screening for the presence of biofilm-associated genes (BAG) (spgM, rmlA and rpfF), their mutational variability, and assessment of the adherent growth on a polystyrene surface. The methodology included: PCR amplification, whole-genome sequencing (WGS) and crystal violet microtiter plate assay for biofilm quantification. The overall incidence of BAG was: spgM 98.6%, rmlA 86%, and rpfF 66.5%. The most prevalent genotype was spgMţ/rmlAţ/rpfFţ (56.1%), followed by spgMţ/rmlAţ/rpfF- (28.5%), and spgMţ/rmlA-/rpfFţ (9.5%), with their significant predominance in lower respiratory tract isolates compared to those with other origin (P < 0.001). All strains examined were characterized as strong biofilm producers (OD550 from 0.224 +- 0.049 to 2.065 +- 0.023) with a single exception that showed a weak biofilm-forming ability (0.177 +- 0.024). No significant differences were observed in the biofilm formation according to the isolation source, as well as among COVID-19 and non-COVID-19 isolates (1.256 +- 0.028 vs. 1.348 +- 0.128, respectively). Also, no correlation was found between the biofilm amounts and the corresponding genotypes. WGS showed that the rmlA accumulated a larger number of variants (0.0086 per base) compared to the other BAG, suggesting no critical role of its product to the biofilm formation. Additionally, two of the isolates were found to harbour class 1 integrons (7-kb and 2.6-kb sized, respectively) containing sul1 in their 30 conservative ends, which confers sulfonamide resistance. To the best of our knowledge, this is the first study on S. maltophilia biofilm formation in Bulgaria, which also identifies novel sequence types (ST819, ST820 and ST826). It demonstrates the complex nature of this adaptive mechanism in the multifactorial pathogenesis of biofilm-associated infections.  Kulcsszavak: Stenotrophomonas maltophilia, biofilm formation, biofilm-associated genes, phenotypic characteristics, PCR screening, whole-genome sequencing