Abstract: Resistance of some bacteria, especially some stains of E. coli to common antimicrobial agents has created an urgent need to develop alternative antimicrobial drugs from herbs that are safe, cheap and may overcome the resistance of the pathogens. The crude aqueous extract of Psidium guajava leaf which is known to possess some antibacterial properties was further subjected to sequential fractionation with organic solvents (chloroform, ethyl acetate, normal butanol) of different polarity. This was done until the organic layer was visibly clear to obtain chloroform, ethyl acetate and n-butanol soluble fractions and residual aqueous fraction. Phytochemical screening and antibacterial activity of organic solvents soluble fractions and residual fraction of the extract on some gram positive and gram negative microbes were carried out. The different fractions showed variation in phytochemical constituency and thus in their antibacterial properties. The ethyl acetate soluble fraction of the extract showed broad spectrum antibacterial properties against all the organisms tested. The fraction also showed a good activity against E. coli at a relatively lower concentration and hence could possibly be use against E. coli infections.