Zinc oxide nanoparticles (ZnO NPs) have anticancer, antidiabetic, antibacterial, and anti-inflammatory properties in the biotechnology field. Cafestol, which is the one of the diterpene in coffee, has antimicrobial and anticancer activities. This study aimed to synthesize the cafestol-chitosan-ZnO NP system to evaluate its antibacterial activity. ZnO NPs were produced by the chemical precipitation method. Optimization studies were performed to obtain the desired size of the ZnO NPs. The type of zinc salt [ZnCl2, Zn(SO4)], salt concentration (0.1; 0.2; 0.5 M), base type (NH3, NaOH), reaction time (6, 12, 18, 24 h), mixing speed (300, 400, 500 rpm), and calcination time (1, 2, 3 h) parameters in the method were investigated to yield the targeted size. The optimum experimental conditions required to synthesize in the 45-60 nm size range were determined as a 0.2 M Zn(SO4) salt type and concentration, NaOH base type, 18 h reaction time, at 400 rpm mixing speed and 2 h calcination time. After synthesizing the ZnO NPs coated with chitosan (CS), the cafestol was ligated to the CS-ZnO NP. It was proved by Fourier-transform infrared, differential scanning calorimeter/thermogravimetry analysis/diamond thermogravimetry analysis and scanning electron microscope analyses that cafestol-CS-ZnO NPs were successfully synthesized. The antibacterial effects of cafestol, CS, ZnO NPs, CS-ZnO NPs and cafestol-CS-ZnO NPs were evaluated on human pathogenic Gram-positive strainsStaphylococcus aureusATCC 25923and Bacillus cereusATCC 11778 and Gram-negative strainsPseudomonas aeruginosaPA01andEscherichia coliATCC 25922. The ZnO NPs, CS-ZnO NP, and cafestol-CS-ZnO NP (varying between 20 and 1000 mu g/mL) completely inhibited bacterial growth for S. aureus, B. cereus, and E. coli. The incorporation of CS and CS-cafestol improved the antibacterial activity of the ZnO NPs samples against P. aeruginosa PA01 with a 75-87.5% inhibition. The obtained data shows that CS-ZnO and cafestol-CS-ZnO NPs have great potential for biological and pharmaceutical applications.