Cefotaxime (CTX) is a third-generation cephalosporin antibiotic with broad-spectrum activity against Gram negative and Gram positive bacteria. However, like other third-generation cephalosporin antibiotics, its efficacy is declining due to the increased prevalence of multidrugresistant (MDR) pathogens. Recent advances in nanotechnology have been projected as a practical approach to combat MDR microorganisms. Therefore, in the current study, gold nanoparticles (AuNPs) were prepared using cefotaxime sodium, which acted as a reducing and capping agent, besides having well-established antibacterial activity. The synthesized cefotaxime-loaded gold nanoparticles (C-AuNPs) were characterized by UV-Visible spectroscopy, FTIR, TEM and DLS. In addition, the in vitro antibacterial activity of C-AuNPs was assessed against both Gram-positive and Gram-negative bacteria. UV-Visible spectroscopy verified the formation of C-AuNPs, while TEM and DLS verified their nano-size. In addition, CTX loading onto AuNPs was confirmed by FTIR. Furthermore, the colloidal stability of the synthesized C-AuNPs was ascribed to the higher net negative surface charge of C-AuNPs. Most importantly, the synthesized C-AuNPs showed superior antibacterial activity and lower minimum inhibitory concentration (MIC) values against Gramnegative (Escherichia coli, Klebsiella oxytoca, Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria, compared with pure CTX. Collectively, CTX was successfully adopted, as reducing and capping agent, to synthesize stable, nano-sized spherical C-AuNPs. Furthermore, loading CTX onto AuNPs could efficiently restore and/or boost the antibacterial activity of CTX against resistant Gram-negative and Gram-positive bacteria.