Chronic wounds represent an important burden on the healthcare system, requiring frequent hospitalizations and expensive treatments. It is now recognized that a primary factor contributing to a non-healing trajectory and a low therapeutic response is the biofilm infection. The purpose of this study was to identify the bacterial isolates collected from chronic skin wounds of hospitalized patients and to evaluate their antimicrobial susceptibility profiles, virulence factors, as well as the ability to develop biofilms in vitro. A number of 44 wound samples were collected from 39 patients. The isolated strains belonged to seven different microbial species, i.e.: Staphylococcus aureus (32 strains), followed by Pseudomonas aeruginosa (4), Escherichia coli (3), Klebsiella pneumoniae (2), Proteus mirabilis (1), Citrobacter freundii (1), group G β-hemolytic streptococci (1). In comparison to the other isolates, P. aeruginosa strains exhibited the highest capacity to develop complex biofilm structures in vitro, followed by S. aureus, with insignificant differences between MRSA and non-methicillin resistant isolates. The Enterobacteriaceae strains expressed less virulent phenotypes, lower adherence to epithelial cells and biofilm forming capacity, but also significant resistance phenotypes with a potential of unfavorable epidemiological outcome. The isolation of MRSA, ESBL-producing microorganisms and multiple antibiotic resistant P. aeruginosa suggests the potential risk of nosocomial spread and the potential severe outcome in case of bacteremia and sepsis. This study represents an important step in elucidating the host-wound microbiome interaction, by describing various resistance and virulence threats of microorganisms colonizing and/or infecting the chronic wounds. However, in order to establish a statistical relevant correlation, larger studies are needed.