Abstract. Escherichia coli is a gram-negative bacilli one of the causative agent of gastrointestinal infections. The decrease of the sensitivity of E.coli to antibiotics has led to the need for alternatives in treatment, including the use of mahogany seeds (Swietenia mahagoni) which are one of the traditional plants that contain flavonoids, alkaloids that antimicrobials. The purpose of this study was to determine the effect of methanol extract of mahogany seeds on the activity of E.coli bacteria. This research is an in vitro laboratory experiment using the well method with six group that is four groups of mahagony seeds methanol extract with concentration of 100%, 75%, 50%, 25% group, gentamicin as the positive control and aquadest as the negative control. The samples were incubated in an incubator at 37°C for 24 hours. The inhibition zone was measured. In this study, there was no average inhibition zone of the methanol extract of mahogany seeds at various concentrations, the positive control was 27.9 mm. It can be concluded that the methanol extract of mahogany seeds is not effective as an antibacterial with low inhibition (resistant). The results of low inhibition might owed to the extraction method, the solvent used, the concentration of the extract which is not large enough, the ecological factors where the plant grows Abstrak. Escherichia coli adalah bakteri basil gram negatif salah satu agen penyebab infeksi gastrointestinal. Terjadinya penurunan persentase kepekaan E.coli terhadap antibiotik menyebabkan perlu adanya alternatif dalam pengobatan, diantaranya melalui pemanfaatan biji mahoni (Swietenia mahagoni) sebagai salah satu tanaman tradisional yang memiliki kandungan flavonoid, alkaloid yang berfungsi sebagai antimikroba. Tujuan penelitian ini adalah untuk mengetahui efek ekstrak metanol biji mahoni terhadap aktivitas bakteri E.coli. Penelitian ini bersifat eksperimental laboratorium secara in vitro menggunakan metode sumuran terdiri dari 6 kelompok perlakuan. Masing-masing kelompok berisi koloni bakteri diberi lubang sumuran yang diinjeksi ekstrak metanol biji mahoni dengan konsentrasi 100%, 75%, 50%, 25%, gentamisin sebagai kontrol positif dan aquadest sebagai kontrol negatif. Sampel diinkubasi dalam suhu 37ºC selama 24 jam. Kemudian dilakukan pengukuran zona hambat. Hasil penelitian ini tidak ditemukan rata-rata zona hambat dari kelompok esktrak metanol biji mahoni pada berbagai konsentrasi, namun pada kontrol positif terdapat zona hambat sebesar 27,9 mm. Berdasarkan hasil yang ditemukan, dapat disimpulkan bahwa ekstrak metanol biji mahoni metode sumuran belum efektif sebagai antibakteri. Hasil daya hambat yang rendah dapat dikarenakan metode ekstraksi, pelarut yang digunakan, konsentrasi ekstrak yang kurang besar, faktor ekologi tempat pertumbuhan tanaman
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