Abstract Background Cerenkov Luminescence Imaging (CLI) is an emerging technology for intraoperative margin assessment. Previous research only evaluated radionuclide 18-Fluorine (¹⁸F); however, for future applications in prostate cancer, 68-Gallium (⁶⁸Ga) seems more suitable, given its higher positron energy. Theoretical calculations predict that ⁶⁸Ga should offer a higher signal-to-noise ratio than ¹⁸F; this is the first experimental confirmation. The aim of this study is to investigate the technical performance of CLI by comparing ⁶⁸Ga to ¹⁸F. Results The linearity of the system, detection limit, spatial resolution, and uniformity were determined with the LightPath imaging system. All experiments were conducted with clinically relevant activity levels in vitro, using dedicated phantoms. For both radionuclides, a linear relationship between the activity concentration and detected light yield was observed (R² = 0.99). ⁶⁸Ga showed approximately 22 times more detectable Cerenkov signal compared to ¹⁸F. The detectable activity concentration after a 120 s exposure time and 2 × 2 binning of ¹⁸F was 23.7 kBq/mL and 1.2 kBq/mL for ⁶⁸Ga. The spatial resolution was 1.31 mm for ¹⁸F and 1.40 mm for ⁶⁸Ga. The coefficient of variance of the uniformity phantom was 0.07 for the central field of view. Conclusion ⁶⁸Ga was superior over ¹⁸F in terms of light yield and minimal detection limit. However, as could be expected, the resolution was 0.1 mm less for ⁶⁸Ga. Given the clinical constraints of an acquisition time less than 120 s and a spatial resolution < 2 mm, CLI for intraoperative margin assessment using ⁶⁸Ga could be feasible.