The persistent challenge of bacterial infections in clinical practice necessitates the development of precise diagnostic tools capable of differentiating infectious and inflammatory processes. This study presents the development and evaluation of [68Ga]Ga-NOTA-UBI 29-41, a novel PET radiotracer designed for specific bacterial infection imaging. The tracer was synthesized through 68Ga-labeling of a NOTA-conjugated ubiquicidin 29-41 derivative, demonstrating excellent radiochemical properties and good in vitro stability in both saline and mouse serum. In vitro binding assays confirmed specific targeting of Staphylococcus aureus, while in vivo studies in mice models revealed significantly higher uptake in infected versus inflamed muscle tissue (p < 0.01). MicroPET imaging further validated the tracer's ability to selectively accumulate at infection sites, with pharmacokinetic analysis showing predominant renal clearance and rapid blood clearance kinetics. These findings collectively demonstrate the potential of [68Ga]Ga-NOTA-UBI 29-41 as a clinically translatable agent for accurate discrimination between bacterial infections and sterile inflammation.