Plant-based biosynthesis of nanomaterials offers a sustainable and eco-friendly alternative compared to the conventional chemical and physical methods. In this study, zinc oxide nanoparticles (ZnO NPs) were synthesized using an aqueous extract of Opuntia ficus-indica (OFI) as a natural reducing and stabilizing agent. TEM and FE-SEM analyses revealed predominantly spherical nanoparticles with smooth surfaces, minimal aggregation, and an average particle size ranging from 34 to 70 nm. XRD patterns confirmed the hexagonal wurtzite crystalline structure with high phase purity, while EDX spectra showed strong zinc and oxygen peaks, verifying elemental composition. FT-IR analysis indicated the presence of hydroxyl, carbonyl, and polysaccharide functional groups from OFI phytochemicals responsible for reduction and stabilization. The ZnO NPs exhibited potent antibacterial and antibiofilm activities against multidrug-resistant ESKAPE pathogens, with inhibition zone diameters of 16–26 mm and minimum inhibitory concentrations (MICs) of 3.125–25 μg/mL, along with biofilm inhibition rates of 20–72.15 %. A synergistic enhancement of antibacterial efficacy was observed when combined of ZnO NPs with conventional antibiotics. The ZnO NPs also showed strong antioxidant activity, achieving up to 80.26 % DPPH radical scavenging at 100 μg/mL. Cytotoxicity assay of ZnO NPs demonstrated dose-dependent anticancer effects against A549 lung carcinoma cells, with substantial cell viability reduction at increasing concentrations. Collectively, these findings highlight the multifunctional therapeutic potential of OFI-mediated ZnO NPs as sustainable antibacterial, antibiofilm, antioxidant, and anticancer agents.