The present research was aimed to investigate the antimicrobial potential of biologically synthesized silver nanoparticles from leaf extract of Eucalyptus globules against certified pathogenic microorganisms Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella, Staphylococcus aureus and also acne inducing bacteria, using agar well diffusion method. Highest antimicrobial activity (20 mm) against Staphylococcus aureus was recorded with methanolic extract of Eucalyptus globules leaves in comparison to other extracts. Phytochemical analysis of crude extract revealed the presence of alkaloids, carbohydrates, flavonoids, saponins, steroids, soluble starch, terpenoids and tannins. The silver nanoparticles synthesized from the methanolic extract using silver nitrate showed peak at 375 nm. The characterization of nanoparticles for size, shape and nature using X-ray diffraction (XRD) and Atomic Force Microscopy (AFM) revealed that the particle size ranged between 15-71 nm. The efficacy of synthesized nanoparticles was checked against four acne inducing isolates and also other certified pathogenic strains. It was found that the overall effectiveness of AgNp’s was much higher than crude extract and also exceptionally high in some cases (i.e. higher than positive control). The AgNp’s were quite effective against the pathogenic strains even at very low concentration (0.0097 μg/100 ml). The results indicated a marked improvement in effectiveness of plant extracts at nanoscale. Further Eucalyptus leaf extract has potential to be used as alternate to synthetic medicines as they are relatively cheaper and safer to use with less or no side effects but only after intensive R&D and clinical trials.