Multidrug-resistant (MDR) bacterial infections, particularly those caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, have substantially compromised the therapeutic efficacy of cefotaxime, a third-generation cephalosporin antibiotic. The present study was aimed at formulating and evaluating cefotaxime-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a controlled-release antibacterial delivery system capable of overcoming the pharmacokinetic and resistance-related limitations of the conventional drug. Nanoparticles were prepared by the solvent evaporation/nanoprecipitation technique, and formulation variables – namely drug-to-polymer ratio, PLGA concentration, surfactant (polyvinyl alcohol, PVA) concentration, stirring speed, and sonication time – were systematically optimized using a one-factor-at-a-time approach. The optimized formulation (drug:polymer 1:5, PLGA 2.0% w/v, PVA 1.5% w/v, stirring speed 14,000 rpm, sonication time 6 min) yielded nanoparticles with a mean particle size of 165.3 ± 1.9 nm, a polydispersity index (PDI) of 0.214 ± 0.02, and a zeta potential of −28.6 ± 1.4 mV. Drug loading and encapsulation efficiency were found to be 21.4 ± 0.3% and 93.8 ± 0.7%, respectively. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) confirmed the absence of significant drug–polymer interaction. Scanning and transmission electron microscopy revealed smooth, spherical, well-dispersed nanoparticles. In vitro release studies demonstrated a biphasic pattern, with an initial burst release followed by sustained release of cefotaxime for up to 72 hours. Antibacterial evaluation by agar well diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time–kill assays revealed significantly enhanced and sustained activity of the nanoformulation against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to free cefotaxime. The formulation remained physicochemically stable for 90 days under International Council for Harmonisation (ICH) long-term storage conditions and exhibited hemolysis below the 5% safety threshold, indicating excellent hemocompatibility. Collectively, these findings indicate that cefotaxime-loaded PLGA nanoparticles represent a promising, biocompatible, and scalable sustained-release platform with potential application in the treatment of MDR bacterial infections. Keywords: Cefotaxime; PLGA nanoparticles; Multidrug resistance; Controlled drug release; Encapsulation efficiency; Antibacterial activity; Nanomedicine