Breast cancer remains a major global health challenge, and understanding interactions between pharmacological agents and radiopharmaceuticals is important for interpreting molecular imaging findings. Glycyrrhizic acid (GA), a bioactive triterpenoid derived from Glycyrrhiza glabra, has reported anti-inflammatory and anticancer activities; however, its effects on PSMA-targeted radiotracer biodistribution remain poorly investigated. This study evaluated GA effects on tumour-related biological responses and [99mTc]Tc-PSMA biodistribution in vitro and in vivo. Cytotoxicity, migration, and invasion were assessed in MDA-MB-231 breast cancer cells. Cell-free PSMA enzyme kinetics and a separate cellular [99mTc]Tc-PSMA uptake assay were performed to distinguish effects on PSMA enzymatic activity from cell-associated radiotracer uptake. In vivo studies used a DMBA-induced mammary tumour model in female Sprague-Dawley rats, followed by SPECT/CT imaging and tissue biodistribution analysis. GA reduced cell viability concentration-dependently (IC₅₀ = 20 µM at 48 h) and suppressed migration and invasion. Cell-free kinetic analysis showed an apparent non-competitive PSMA inhibition pattern (Ki = 12.4 ± 1.5 µM), although this does not establish the molecular binding site. In vivo, GA reduced tumour uptake of [99mTc]Tc-PSMA by 57% (P