The current work seeks to analyze TLC profile, phytochemical constituents, and antimicrobial activities of Eucalyptus globulus root extract. Eucalyptus globulus including other medicinal plants are sources of bioactive compounds; however, the root extracts of this species are still unknown. In order to close this gap, a methanolic extract of the roots was prepared by the use of soxhlet extraction technique giving a percentage yield of 7.23% w/w which could be considered high since it suggests good recovery of phytoconstituents have been made. Phytochemical analysis of the extract call for presence of several bio active compounds among them are flavonoids, terpenes, phenol, tannin, saponin and glycoside that possess various pharmacological activities. TLC profiling for flavonoids had an Rf value of 0.87 while terpenes had an Rf value of 0.31, phenols had an Rf of 0.25 and tannins had Rf of 0.19. These findings agreed with the chemical variation and abundance of the extract. Apart from the phytochemical characterization, the ant-bacterial efficiency of the extract was also assessed against different bacterial and fungal isolates. The extract showed moderate antibacterial efficacy against the examined bacteria especially gram-positive bacteria S. aureus and K. pneumoniae with MIC values comparable to the standard Gentamycin. Still, the activity documented against the gram-negative bacteria and the fungal strains was relatively low. However, the inhibition zones noticed during the antifungal assays indicate that extract has the ability to cure fungal infections. The outcome of this research disproves the use of Eucalyptus globulus root to give phytochemicals used in antimicrobial products. On one hand, its moderate antifungal potency, however, demonstrate its SOME limitation, but on the other, the extract exhibit activity against gram-positive bacteria making it a potential natural source for manufacture of efficient antimicrobial compounds. Subsequent research works on the identification and profiling of these molecules may significantly improve the therapeutic value of the plant in creating new forms of herbal medicine and antimicrobial pharmaceuticals.