Phenethyl Isothiocyanate (PEITC) has good pharmacological activities in treating many diseases. However, its application has been limited due to its high hydrophobicity, low dissolution profile, storage stability difficulties, and rapid clearance. The current study aimed to make PEITC-loaded biocompatible low-toxic nanoparticles (NPs) and evaluate their physicochemical features and anti-bacterial potential. BSA and PEG were utilized as biodegradable polymers to fabricate NPs by emulsification procedure, which was then optimized using the Box-Behnken design (BBD). The fabricated optimized PEITC NPs (Run: 12) showed lower mean particle size (185.6 ± 0.8nm), good polydispersity index (0.22 ± 0.02), and Zeta potential (−23.3 ± 0.5mV), with higher entrapment efficiency (94.36 ± 0.16%) and drug loading (80.14 ± 0.13%) suggesting higher colloidal stability and a larger amount of drug can be delivered at the target site. Drug-loaded BSA-PEG NPs showed a prolonged release of PEITC (89.72% in 24 h) from the BSA matrix following the Higuchi model (R2 value = 0.987). The optimized NPs and the free drug were further screened for anti-bacterial activity, where NPs exhibited more potent anti-bacterial activity than free PEITC against all four bacterial strains by presenting higher killing efficiency. In contrast, free PEITC elicited bacteriostatic activity only. Of all the strains, S. aureus alone was more susceptible to drug-loaded NPs/free drugs. These convincing results helped to further exploration for in vivo anti-bacterial activity. Prior to the in vivo bacterial eradication studies, the doses of NPs administered to the animals are ensured for safety and biocompatibility in both in vitro and in vivo studies. Finally, in a S. aureus-induced mice infection model, PEITC NPs showed a very significant decrease in the area of infection (n=4, p < 0.05) than the control, blank NPs and free drug-treated animal groups. The PEITC NPs decreased the bacterial infection to almost negligible, with approximately 15.2 folds decreased bacterial viability than the free drug.