Propolis, a natural bee-derived product rich in diverse phytochemicals with potential therapeutic benefits, remains underexplored in Algeria. This study investigated the molecular profile, antioxidant capacity, and antibacterial activity of propolis sourced from two bioclimatically distinct Algerian regions (humid subtropical Batna and hot desert Biskra) using electrospray ionization mass spectrometry, ultra-high-performance liquid chromatography with diode array detection, and gas chromatography–mass spectrometry. Significant regional variations were observed, with propolis extract 2 (PE2) exhibiting a higher bioactive content, including a constituent not previously reported in propolis. Antioxidant assays (2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), ferric reducing antioxidant power, and phenanthroline) demonstrated that PE2 consistently outperformed propolis extract 1 and the reference standards (DPPH IC₅₀: 27.74 µg/mL; FRAP: 5.16 µg/mL). Antibacterial testing demonstrated potent bactericidal effects, particularly for PE2, with minimum inhibitory concentration values equivalent to the minimum bactericidal concentrations required against Staphylococcus aureus ATCC 25923 (18.75 µg/mL) and Escherichia coli ATCC 25922 (133 µg/mL). Molecular docking identified nine bioactive compounds with high KEAP1 binding affinity, with 1,3-O-caffeoyl-dihydrocaffeoylglycerol (first time reported in propolis) showing the strongest binding affinity (−11.02 Kcal/mol). In silico pharmacokinetic predictions further verified its drug-like properties. These findings suggest the tested Algerian propolis samples, as a source of natural alternative antioxidants and antimicrobials, provide a basis for future research in drug discovery and development.