ABSTRAK Uji ekstrak metanol jamur tiram (Pleurotus ostreatus) terhadap pertumbuhan bakteri Staphylococcus aureus dan Escherichia coli telah dilakukan dengan tujuan untuk mengetahui aktivitas ekstrak metanol jamur tiram dalam menghambat pertumbuhan bakteri tersebut. Ekstraksi jamur tiram dilakukan dengan menggunakan metode maserasi, sedangkan pengujian aktivitas antibakteri dilakukan dengan metode difusi kertas cakram (Kirby-Bauer) dengan variasi konsentrasi ekstrak yaitu 5; 10; 20 dan 35%. Hasil karakterisasi ekstrak diperoleh kadar sari sebesar 3,75%, kadar abu total 1,60%, kadar sari larut air 30%, dan kadar sari larut etanol 60%. Skrining fitokimia menunjukkan bahwa ekstrak metanol jamur tiram mengandung metabolit sekunder berupa senyawa alkaloid, saponin dan steroid. Hasil uji aktivitas antibakteri menunjukkan ekstrak metanol jamur tiram memiliki zona hambat terbesar terhadap bakteri S. aureus pada konsentrasi 20% yaitu 13,48 mm dan zona hambat terbesar terhadap bakteri E. coli pada konsentrasi 35% yaitu 13,03 mm. Hal ini menunjukkan bahwa ekstrak metanol jamur tiram memiliki aktivitas antibakteri terhadap S. aureus dan E. coli Kata kunci: Ekstrak metanol Pleurotus ostreatus, Staphylococcus aureus, Escherichia coli. ABSTRACT Methanol extract test from oyster mushroom (Pleurotus ostreatus) towards bacterial of Staphylococcus aureus and Escherichia coli has been conducted to know the activity of methanol exstract from oyster mushroom in inhibiting the growth of those bacteria. Extraction from oyster mushroom was conducted using a maceration method, while testing of antibacterial activity was conducted using a disc paper diffusion method (Kirby-Bauer) with variation of extract concentrations of 5; 10; 20 and 35%. The results of extract concentrations obatained water content of 3,75%, total ash content of 1,60%, water soluble extract content of 30% and ethanol soluble extract content of 60%. Phytochemical screening of methanol extract of oyster mushrooms produces secondary metabolites in the from alkaloid compounds, saponins, and steroids. The results of antibacterial activity test showed that methanol extract of oyster mushroom has the largest inhibitory zone to S. aureus bacteria was shown at 20% concentration with 13,48 mm diameter and the largest inhibitory zone to E. coli bacteria was shown at 35% concentration with 13,03 mm diameter. This shows that the methanol extract of oyster mushrooms has antibacterial activity against S. aureus and E. coli. Keywords: Methanol extract Pleurotus ostreatus, Staphylococcus aureus, Escherichia coli.