The antimicrobial activity against four pathogenic bacteria and two fungi was examined in the methanolic extracts of Agave species using disk diffusion method. Among the three examined species; A.americana, A.americana var. marginata and A.angustifolia var. marginata, the extract of A.americana demonstrated the broadest antimicrobial activity with an inhibition zone ranging from 10-19 mm. MIC (Minimum Inhibitory Concentration) of this plant showed a value of 9 mg/ml against Candida albicans. However, A.angustifolia var. marginata extract showed the weakest antibacterial (11-12 mm) and antifungal (0-9 mm) activities. Another three Agave species (A. desmettiana, A.pygmae and A.ferox) were added to the aforementioned species to carry out a genetic variability study using Start Codon Targeted (SCoT) and Inter Simple Sequence Repeat (ISSR) markers. The percentage of polymorphism was more than 60% for the two markers, however SCoT markers showed higher percentage of polymorphic bands (62.85%) than the ISSR (57.14%). High level of polymorphism and a wide range of similarity values between accessions were demonstrated, with the highest similarity (1.00) between A.angustifolia var. marginata and A. desmettiana. The dendrogram separated the examined species into two main clusters. Our present study suggests using ISSR and SCOT markers for successful authentication of these Agave species.