| Summary: | <i>Annona glabra</i> Linn is employed in conventional medicine to treat a number of human disorders, including cancer and viruses. In the present investigation, the significant phytochemical components of <i>Annona glabra</i> hexane extract were identified using gas chromatography–mass spectrometry (GC-MS) analysis. Three major compounds were identified in the hexane extract: tritriacontane (30.23%), 13, 17-dimethyl-tritriacontane (22.44%), and limonene (18.97%). MTT assay was used to assess the cytotoxicity of the extract on six human cancer cell lines including liver (HepG-2), pancreas (PANC-1), lung (A-549), breast (MCF-7, HTB-22), prostate (PC-3), and colon (CACO-2, ATB-37). The extract exhibited significant cytotoxic activity against both CACO-2 and A-549 cancer cell lines (IC<sub>50</sub> = 47 ± 0.74 μg/mL and 56.82 ± 0.92 μg/mL) in comparison with doxorubicin (IC<sub>50</sub> = 31.91 ± 0.81 μg/mL and 23.39 ± 0.43 μg/mL) and of SI of 3.8 and 3.1, respectively. It also induced moderate-to-weak activities against the other cancerous cell lines: PC-3, PANC-1, MCF-7, and HepG-2 (IC<sub>50</sub> = 81.86 ± 3.26, 57.34 ± 0.77, 80.31 ± 4.13, and 57.01 ± 0.85 μg/mL) in comparison to doxorubicin (IC<sub>50</sub> = 32.9 ± 1.74, 19.07 ± 0.2, 15.48 ± 0.84 and 5.4 ± 0.22 μg/mL, respectively) and SI of 2.2, 3.1, 2.2, and 3.1, respectively. In vitro anti-HSV1 (Herpes simplex 1 virus) and HAV (Hepatitis A virus) activity was evaluated using MTT colorimetric assay with three different protocols to test protective, anti-replicative, and anti-infective antiviral activities, and three separate replications of each experiment were conducted. The plant extract showed promising protective and virucidal activity against HSV1 with no significant difference with acyclovir (79.55 ± 1.67 vs. 68.44 ± 7.62 and 70.91 ± 7.02 vs. 83.76 ± 5.67), while it showed mild protective antiviral activity against HAV (48.08 ±3.46) with no significant difference vs. acyclovir (36.89 ± 6.61). The selected main compounds were examined for their bioactivity through in silico molecular docking, which exhibited that limonene could possess the strongest antiviral properties. These findings support <i>Annona glabra’s</i> conventional use, which is an effective source of antiviral and anticancer substances that could be used in pharmaceuticals.
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