Aims: The aim of the study was to examine the effect of different process parameters which including; initial juglone amount, initial poly(d,l-lactide co-glycolide) amount, polyvinyl alcohol volume and polyvinyl alcohol concentration on encapsulation of juglone to poly(d,l-lactide co-glycolide) nanoparticles. Methods and Results: The synthesized nanoparticle formulations were analyzed for reaction yield, encapsulation efficiency, particle size, polydispersity, zeta potential and juglone release. In conjunction with the highest encapsulation rate, the highest amount of juglone release was obtained for F4 formulation, which has 281.8 nm particle size, 0.217 polydispersity index, and -19.55 mV zeta potential. After the detailed physicochemical characterization of this formulation, the four different kinetic models were used and it was found that juglone release mechanism controlled by Fickian diffusion method. According to antimicrobial activity results, minimal inhibitory concentration (MIC) values of both F4 and free juglone is higher for Gram negative bacteria than Gram positive bacteria. Inhibition zone diameters in the quantitative methods are found 15 and 16 mm for Staphylococcus aureus, 9 and 7 mm for Bacillus cereus, respectively, for F4 and free juglone. Moreover, the MIC values for qualitative methods were found 31.5 mu g ml(-1) for two bacteria strains. Conclusions: It was found that the antibacterial activity of the juglone nanoparticles was higher and longer than the free juglone. Additionally, a similar antimicrobial effect with a lower juglone amount (obtained from controlled release study) indicates that nanoparticle formulation is more effective. Significance and Impact of the Study: The use of nanoparticle formulations of juglone in biological systems and applications could be more beneficial than its free form due to its toxicity.