Infections with Salmonella species are among the major foodborne outbreaks of concern globally. This study aimed to characterize virulence determinants, antibiotic susceptibilities and biofilm formation ability of 29 Salmonella spp. isolated from chicken and human sources in Egypt. Molecular identification of virulence-associated genes revealed the detection of avrA gene in 100% of the examined isolates. The sopB, stn, hilA and bcfC genes were identified in 91.3% of the isolates, while, 86.2% and 31.03% harbored mgtC gene and spvC genes, respectively. The pefA and fimH genes were only identified in three isolates (10.3%). Fifteen antimicrobials were chosen to assess the susceptibility of the isolates to these drugs. The majority (31.03%) was resistant to 10 antibiotics; meanwhile, 89.6% were resistant to at least 5 antimicrobials. Out of 29 Salmonella isolates, 89.76%, 82.8% and 37.9% were biofilm producers at 35°C, 25°C, and 4°C, respectively. Significant correlation was observed between different Salmonella serotypes and their ability to produce biofilms. In conclusion, frequent monitoring of virulence determinants, antibiotic susceptibility and biofilm characteristics of Salmonella spp. is essential to improve food safety.