Psidium guineense SW., commonly known as sour guava, is used in traditional medicine to address diverse health issues, such as inflammation, infections, and gastrointestinal disorders. Although previous research has highlighted the antimicrobial properties of the root extract and the antioxidant potential of its fruits, information regarding the activities associated with the leaf extract is lacking. The aim of the study was to use the traditional knowledge of the biological properties of P. guineense and investigate its antimicrobial activity, mechanism of action, synergistic effects, and antivirulence activity against Staphylococcus aureus. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined for S. aureus, including antibiotic-resistant isolates. The synergy between the extract and antibiotics was evaluated. A growth curve was constructed for S. aureus treated with the extract. The anti-hemolytic activity was assessed by inhibiting hemolysis in human blood, and the inhibition of staphyloxanthin (STX) was examined. The extract effectively inhibited all strains with MIC and MBC values ranging from 256 to 512 µg/mL and 512 to 1024 µg/mL, respectively. Antimicrobial synergy experiments demonstrated the enhanced effectiveness of antibiotics, especially ciprofloxacin, when combined with the P. guineense extract, especially with ciprofloxacin, resulting in complete synergy against all S. aureus strains. In growth curve tests, the extract inhibited growth at MIC of 256 µg/mL within 8 h and significantly delayed it at MIC/2 (128 µg/mL) within 12 h. In antivirulence tests, the extract effectively reduced hemolysis and STX production by S. aureus at MIC and MIC/2, indicating its potential as an antimicrobial agent with antivirulence properties. The extract effectively inhibited S. aureus, including drug-resistant strains with low MIC and MBC values, exhibiting strong antimicrobial and synergistic properties with antibiotics. also In addition, the extract accelerated recovery by limiting bacterial growth and exhibiting antivirulence capabilities, reducing the severe symptoms of S. aureus infections.