In this Research work, Silver Nanoparticles were green synthesized from Psidium guajava leaves and different Characterization techniques including UV-Visible, FT-IR, SEM and XRD were all employed to ascertain the absorption peaks, functional group, surface morphology and crystalline size of the nanoparticles respectively. These nanoparticles green synthesized were applied against four different pathogens namely, S. aureus (gram- positive bacteria), E. coli (gram- negative bacteria), C. albicans (gram- positive fungus) and Aspergillus niger and the investigation showed that the Silver nanoparticles synthesized were potent against the selected pathogens. From the UV-Vis spectral analysis, it was observed that highest absorption peaks appeared at 400nm and 500nm reflecting the surface plasmon resonance of silver Nanoparticles from guava leaves which is characteristic of Silver Nanoparticles. From the FT-IR spectrum of the sample under studied, the peaks 3416.85 cm-1, 2923.51 cm-1, 1618.95 cm-1, 1384.49 cm-1 and 1033.63 cm-1 were observed where the absorption band at 3416.85 cm-1 corresponds to the stretching due to N-H, while the band at 2923.51 cm-1 is associated with C-H stretch of alkane and O-H stretching. The peak at 1618.95 cm-1 shows C=C stretching, 1384.49 cm-1 reveals the existence of C-H bending and 1033.63 cm-1 depicted C-O stretching. The SEM analysis revealed the shape of the nanoparticles as being spherical while XRD result admits that the average size of the green synthesized Ag NPs was 45.5 nm using the Scherer’s formula. Augmentin was used as control at concentration of 300μg/L throughout antimicrobial studies. Different concentrations of 100, 200, 300, 400 and 500μg/L of Silver Nanoparticles were tested against each pathogen. It was discovered that with increase in concentrations of Silver Nanoparticles of all the pathogens, there generally appeared to be increase in inhibition zone. At higher concentration of 500μg/L, the zones of inhibition were in the following order; 22.50 mm, 17.00mm, 15.44mm, and 13.23mm for E. Coli, S. aureus, C. albicans and Aspergillus niger respectively. For each concentration investigated, E. coli, demonstrated higher zone of inhibition as opposed to all other pathogens investigated in this research.