Pancratium maritimum is a species of bulbous plant that belongs to the Amaryllidaceae family, it is among one of the most important alkaloid-containing plant families. This study aimed to investigate the antioxidant, antibacterial, and antifungal activities of Pancratium maritimum extracts and their UHPLC-MS analysis. The total phenolic, tannins, and flavonoid content of Tunisian Pancratium maritimum aerial part methanolic extract were determined by colorimetric method. The phytochemicals in P.maritimum methanolic were identified using the UHPLC-MS method. The antioxidant activity was assessed using a DPPH assay. The antibacterial and antifungal properties were evaluated against a diverse array of pertinent pathogens using the broth dilution technique. This study showed the presence of alcaloids in methanolic extract as hordenine, lycorine and galanthamine, a strong antioxidant activity was recorded for P.maritimum methanolic extract with a DPPH inhibition of (86.7%). The alkaloid fraction exhibited the most potent antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis bacteria, with minimum inhibitory concentration (MIC) values ranging from 1.125 to 2 mg/mL. Furthermore, it demonstrated significant anticandidal activity against Candida glabrata and Candida parapsilosis, with MIC values ranging from 0.031 to 0.125 mg/mL. P.maritimum extracts have the potential to be utilized for controlling bacterial biofilms in food and food-related situations.