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Copper-based nanoparticles possess broad-spectrum antibacterial activity against both gram-positive and gram-negative bacteria, making them a cost-effective alternative to other metal-based nanoparticles. The development of eco-friendly copper based nanopaticles using biodegradable and non-toxic biosurfactants, such as rhamnolipid is being explored in this study. In the present study, Cu(I)-rhamnolipid nanoparticles (Cu(I)-Rl Nps) was prepared by coprecipitation method. The structural analysis by using FTIR and XRD techniques revealed that Cu(I)-Rl Nps was successfully produced, as indicated by the detectable of ionic and covalent-coordinations bond between rhamnolipid and Cu(I) ions. Further analysis using TEM, PSA and ZPA suggest that the resulted Cu(I)-Rl Nps have spherical shape with the diameter range of 141.7–536.3 nm and the surface charge of −30 mV, respectively. The antibacterial activity of Cu(I)-Rl Nps surpassed that of the copper-based nanoparticles, free-state Cu(I) ions and rhamnolipid, which was determined by MIC/MBC methods. The Cu(I)-Rl Nps inhibition to the growth of Bacillus subtilis ATCC 6633 (Gram-positive) gave the MIC/MBC values of 19/19 μg/mL, while the copper-based nanoparticles, free-state Cu(I) ions and rhamnolipid gave the MIC/MBC value of 1250/2500, 1250/1250, 62/62 μg/mL, respectively. Further test on Escherichia coli ATCC 6538 (Gram-negative) showed that the Cu(I)-Rl Nps gave the MIC/MBC value of 78/78 μg/mL, while the copper-based nanoparticles, free-state Cu(I) ions and rhamnolipid gave the MIC/MBC value of 2500/2500, 2500/2500, 2000/2000 μg/mL, respectively. The increased antibacterial activity of Cu(I)-Rl Nps was due to the synergistic effects between Cu(I) and rhamnolipid.