The approach of sequencing or genotyping to characterize the pathogenic potential of staphylococci from orthopedic device-related infection (ODRI) has been applied in recent studies. These studies described the genomic carriage of virulence in clinical strains and compared it with those in commensal strains. Only a few studies have directly correlated genomic profiles to patient outcome and phenotypic virulence properties in periprosthetic joint infections (PJIs). We investigated the association between genomic variations and virulence-associated phenotypes (biofilm-forming ability and antimicrobial resistance) in 111 staphylococcal strains isolated from patients with PJI and the infection outcome (resolved/unresolved). The presence of a strong biofilm phenotype in Staphylococcus aureus and an antibiotic-resistant phenotype in Staphylococcus epidermidis were both associated with treatment failure of PJI. In S. epidermidis, multidrug resistance (MDR) and resistance to rifampicin were associated with unresolved infection. Sequence type 45 (ST45) and ST2 were particularly enriched in S. aureus and S. epidermidis, respectively. S. epidermidis ST2 caused the majority of relapses and was associated with MDR and strong biofilm production, whereas ST215 correlated with MDR and non/weak biofilm production. S. aureus agr II correlated with resolved infection, while S. epidermidis agr I was associated with strong biofilm production and agr III with non/weak production. Collectively, our results highlight the importance of careful genomic and phenotypic characterization to anticipate the probability of the strain causing treatment failure in PJI. Due to the high rate of resistant S. epidermidis strains identified, this study provides evidence that the current recommended treatment of rifampicin and a fluoroquinolone should not be administered without knowledge of the resistance pattern. IMPORTANCE This study addresses the presence and frequency of particular genetic variants and virulence factors found in staphylococcal bacteria causing periprosthetic joint infection (PJI) of the hip and knee to ascertain their clinical relevance as predictors of treatment failure. We characterized the genetic virulence traits of a large collection of clinical staphylococci isolated from patients with PJI and evaluated their association with the patient's infection outcome. The results showed that S. aureus strains that produced strong biofilms and S. epidermidis strains with resistance to several antibiotics associated significantly with unresolved infection. Some particular genetic variants associated with biofilm formation and multidrug resistance. These traits should be considered important risk factors for the diagnosis and treatment guidance in PJI.