It is known that the phytochemical identification and assessment of biological effects caused by the constituent speciesPsidium cattleianum, which belongs to family Myrtaceae, are poorly held in the literature. The aim of the current study is to investigate the composition of secondary metabolites, the toxicity, and the antimicrobial activity ofP. cattleianumleaves. The crude ethanolic extract of the plant was obtained through maceration and fractionated with hexane, dichloromethane, and ethyl acetate. The crude ethanol extract and the fractions were subjected to phytochemical screening and tested against the microcrustaceanArtemia salinafor toxicological assessment. Antimicrobial tests with crude ethanol extract and the fractions were carried out through the agar diffusion method using broth microdilution againstStaphylococcus aureus,S. epidermidis,Burkholderia cepacia,andEscherichia colistrains. A variety of secondary metabolite groups such as catechins, steroids, phenolic compounds, flavonoids, and saponins was detected. Regarding toxicity, hexane and dichloromethane fractions were considered nontoxic, whereas the crude ethanol extract and the ethyl acetate fraction showed low toxicity. The crude ethanol extract and the fractions, except for the hexane fraction, showed activity against the tested strains. Therefore, the composition of the secondary metabolites, the low toxicity, and the antimicrobial activity suggest that this species is promising in the search and development of new drugs.