Genetic diversity is crucial for understanding the various characteristics of plants, such as their appearance, function, chemical structure, and genetic composition. This study investigated five different basil cultivars, lemon, sweet, Italian, white, and red French, for genetic diversity by ISSR and SCOT analysis and its effect on essential oils' yield and antioxidant, antiviral, anticancer, and antimicrobial activities. The genetic diversity of five basil cultivars was determined using ISSR and SCOT-PCR molecular markers. A total of 60 loci were produced by the ISSR-PCR reactions, 26 of which were polymorphic, while 16 were unique. The level of polymorphism varied from 55.65 to 88.89 %. The molecular profile generated by SCOT-PCR revealed 54 loci, 26 of which were polymorphic, while eight were unique. The level of polymorphism ranged from 50 to 72.72%. The most unique bands were found in the BLe cultivar, 11 by the ISSR markers and 7 by the SCOT markers. The molecular results influence the biological activities of basil cultivars. The GC/MS detected twenty-four compounds; Linalool and methyl cinnamate were the main VOC compounds in the basil profile. The lemon basil essential oil (Ble) had the highest phenolic content (368 mg/g) compared to the other cultivars' EOs; therefore, it exhibited the highest scavenging activity, reducing 89 % of DPPH radicals followed by local basil (Bl). The Ble showed considerable antiviral activity against the bovine viral diarrhea virus (BVDV) as a model for the hepatitis C virus (HCV). Ble's cytotoxic concentration 50% (CC50) was 1400 μg/ml, demonstrating the best antiviral efficacy. Additionally, Ble and Bl exhibited significant antimicrobial activity against pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and pathogenic fungi, i.e., Fusarium oxysporium, Aspergillus niger, Penicillium chrysogenum, Helminthosporium solani, Alternaria alternata, Pythium aphanidermatum, Botrytis cinerea, and Rhizoctonia solani). The anticancer activity of basil EOs was examined against MCF7 cancer cell lines; BLe cultivar reduced 85 % of the viability of MCF7 cancer cells. The examined basil EOs exhibit strong phyto-medical potential based on their chemical structure and antioxidant, antiviral, antimicrobial, and anticancer properties. Based on the molecular and biochemical analysis results, the BLe and Bl cultivars was the best for medicinal use future breeding strategies.