This study aimed to estimate the effect of cobalt nanoparticles (Co NPs) with different concentrations against multidrug-resistant (MDR) pathogenic bacteria. Three isolates of Staphylococcus aureus (gram-positive), Proteus spp. (gram-negative), and Escherichia coli (gram-negative) bacteria were extracted from various clinical examples utilizing routine methods on bacteriological culture media. The antibacterial sensitivity of commercial antibiotics such as Ciprofloxacin, Cefotaxime, Gentamycin, and Amoxicillin was broken down on a Muller Hinton agar plate and evaluated using the disk diffusion method. The study results demonstrated the antibacterial effect of the Co NPs against the bacterial isolates with three different concentrations utilized in the study. The results indicated that the Co NPs showed the highest antibacterial activity when utilizing 100 μg/ml against Escherichia coli followed by Proteus spp and Staphylococcus aureus with zones of inhibition measured as 22.2±0.1 mm, 20.3±0.15 mm, and 15.8±0.1 mm; respectively. Co NPs at a 100 μg/mL concentration showed higher inhibition zones than several common antibiotics except for Ciprofloxacin, which demonstrated better antibacterial activity against the bacterial isolates employed in this study. Scanning Electron Microscope (SEM)and X-Ray diffraction (XRD)studies confirmed that Cobalt nanoparticles (Co NPs) were synthesized from cobalt sulphate solution with a size ranging from 40 nm to 60 nm. The nanoparticles showed a crystalline structure with a round shape and smooth surface. The antibacterial resistance of Co NPs against three common bacteria such as Staphylococcus aureus, Proteus spp, and Escherichia coli was assessed in this study. The optimum concentration of the Co NPs was identified as 100 μg/ml, which could provide a similar or higher antibacterial effect.