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This study was designed to identify the phytochemical constituents and in vitro antimicrobial potential of methanolic extract of Psidiumguajava Linn (Myrtaceae). The phytochemical screening Psidiumguajava leaves showed that the presence of saponins, flavonoids, polyphenol, triterpenoids steroids, alkaloids,carbohydrate, protein, anthroquinone, terpenoids and glycosides whereas alkaloids and phlopatannins was absent in methanol extract. The inhibitory effect of methanolic extract of P. guajava was tested against five bacterial and three fungal strains by using the paper disc diffusion method. Antimicrobial activity was directly proportional to the concentration of Psidiumguajava extract. The Psidiumguajava extract shows highest antimicrobial activity was observed against bacteria when compared with fungi. The high doses (150µl) of Psidiumguajava extract possess similar activity to standard drug as chloramphenicol for bacteria and Fluconazole for fungi. This study is the first scientific report that provides convincing phytochemicals and antimicrobial activity evidence for the relevance of Psidiumguajava leaves thus providing scientific validity to its traditional consumption by the local populace of south India. Psidiumguajava leaves extract had a good potential for therapeutic use against the bacterial and fungal pathogens.