Abstract Background <p>Linezolid (LNZ) is considered one of the last-resort antimicrobial agents reserved for treating methicillin resistant <i>Staphylococcus aureus</i> (MRSA) and vancomycin-resistant <i>Staphylococcus aureus</i> (VRSA). The development of resistance against linezolid necessitates the exploration of novel therapies.&#xa0; Aim: This study aims to investigate the synergistic activity of various combinations of linezolid with non-antibiotic through in vitro and in vivo approaches.</p> Methods and Results <p>In our research, 44 <i>S. aureus</i> isolates were obtained from various clinical sources. <i>S. aureus</i> isolates presented high levels of resistance to β-lactams and moderate resistance to doxycycline and erythromycin. Among the isolates, 43 (97.73%) were MRSA, 10 (22.73%) were linezolid resistant <i>S. aureus</i> (LRSA), and 17 (38.64%) were classified as VRSA. A total of 97.73% of the isolates presented the <i>mecA</i> gene (MRSA), whereas the <i>optrA</i> gene was detected in 9.09% of the isolates (LRSA). &#xa0;The synergistic activity of nine compounds with linezolid was assessed in vitro against LRSA isolates using broth microdilution and checkerboard microdilution methods. Linezolid/cyclizine and linezolid/piroxicam combinations showed fractional inhibitory concentration indexes (FICIs) ranging from 0.28 to 0.5 against LRSA isolates. Time-kill curves were used to confirm their bactericidal activity. Promising combinations (linezolid/cyclizine and linezolid/piroxicam) were further evaluated in vivo LRSA-induced lung infection murine animal model. Compared with monotherapy, combination therapies significantly enhance bacterial eradication and increase sensitivity to linezolid, resulting in superior bacterial eradication. Linezolid/cyclizine and linezolid/piroxicam combinations provided complete protection (100% survival), improved lung pathology, and enhanced clinical scores.&#xa0;</p> Conclusion <p>This study presents two novel combination therapies (linezolid/cyclizine and linezolid/piroxicam) with promising applications in treating severe LRSA infections.</p> Key points <p><UnorderedList Mark="Bullet"> <ItemContent> <p><i>optrA gene was detected in four linezolid S. aureus–resistant isolates (LRSA)</i></p> </ItemContent> <ItemContent> <p><i>Linezolid/cyclizine and linezolid/piroxicam synergism was detected against LRSA.</i></p> </ItemContent> <ItemContent> <p><i>Combinations revealed complete lung protection in lung-infected LRSA murine model.</i></p> </ItemContent> </UnorderedList></p>