This study investigates the phytochemical profile and bioactivities of an ethanolic guava leaves extract (GLE), emphasizing its potential therapeutic applications. The extract yielded 13.73 ± 0.26 g with high amount of total phenolics (44.95 ± 0.21 mg GAE/g) and flavonoids (33.97 ± 0.42 mg QE/g). HPLC analysis identified gallic acid as the dominant phe nolic compound, along with other bioactive constituents such as caffeic acid, naringenin, and catechin, underscoring GLE’s rich antioxidant potential. Antioxidant assays (DPPH, β-carotene-linoleic acid, FRAP) confirmed strong radical scavenging and reducing power, often comparable or superior to gallic acid and TBHQ. Cytotoxicity studies showed selective toxic ity toward A549 lung cancer cells (IC₅₀ = 61.09 µg/mL; selectivity index (SI) = 2.21), with minimal effects on normal Vero cells at low concentrations. GLE induced apoptosis by modulating Bax and Bcl-2 gene expression, particularly in A549 cells. Additionally, GLE demonstrated dose-dependent antiviral activity against HSV-2, achieving up to 76% inhibition, and exhibited moderate antibacterial activity, especially versus K. pneumoniae and E. coli. The findings suggest that GLE may serve as a promising natural source of antioxidants and bioactive agents with selective anti-cancer, antiviral, and antibacte rial properties. Its broad-spectrum efficacy and low toxicity at therapeutic concentrations support further exploration for pharmaceutical and nutraceutical applications.