In exploring the impact of γ-radiation on the biochemical makeup and antimicrobial attributes of Zubaidi truffle (Terfezia boudieri), this study delved into the nuanced interplay between irradiation doses, chemical components, and antimicrobial efficacy. This research scrutinized the influence of γ-radiation on the chemical constitutes and antimicrobial characteristics of Zubaidi truffle (Terfezia boudieri). By applying four distinct doses of γ-radiation (2.5, 5.0, 7.5, and 10 kGy), the study assesses various parameters, including phenols, flavonoids, total and reduced soluble sugars, crude and soluble protein, total amino acids, antioxidant activity, and the susceptibility of examined strains to irradiated and non-irradiated truffle extracts in comparison to standard antibiotics. The findings elucidate that γ-irradiation induces moderate adjustments in the chemical composition, coupled with a dose-dependent escalation in antioxidant activity. Remarkably, irradiated truffle extracts highlight antibiotic efficacy comparable to standard antibiotics, unveiling a nuanced correlation influenced by both radiation dosage and bacterial strain.