The high antibiotic tolerance of Staphylococcus aureus biofilms is associated with challenges for treating periprosthetic joint infection. The toxin-antitoxin system, YefM-YoeB, is thought to be a regulator for antibiotic tolerance, but its physiological role is unknown. The objective of this study was to determine the biofilm and antibiotic susceptibility phenotypes associated with S. aureus yoeB homologs. We hypothesized the toxin-antitoxin yoeB homologs contribute to biofilm formation and antibiotic susceptibility. Disruption of yoeB1 and yoeB2 resulted in decreased biofilm formation in comparison to Newman and JE2 wild-type (WT) S. aureus strains. In comparison to yoeB mutants, both Newman and JE2 WT strains had higher polysaccharide intercellular adhesin (PIA) production. Treatment with sodium metaperiodate increased biofilm formation in Newman WT, indicating biofilm formation may be increased under conditions of oxidative stress. DNase I treatment decreased biofilm formation in Newman WT but not in the absence of yoeB1 or yoeB2. Additionally, WT strains had a higher extracellular DNA (eDNA) content in comparison to yoeB mutants but no differences in biofilm protein content. Moreover, loss of yoeB1 and yoeB2 decreased biofilm survival in both Newman and JE2 strains. Finally, in a neutropenic mouse abscess model, deletion of yoeB1 and yoeB2 resulted in reduced bacterial burden. In conclusion, our data suggest that yoeB1 and yoeB2 are associated with S. aureus planktonic growth, extracellular dependent biofilm formation, antibiotic tolerance, and virulence.Copyright: © 2021 Xinyu Qi et al.