Onion (Allium cepa L) is one type of vegetables which is grown world-wide and form an important part of many diets. But this crop is prone to postharvest loss due to spoilage bacteria. Even though different studies have been done to maximize the onion production in Bahir Dar, postharvest loss of onion was not studied for the presence of microorganism causative agents. Study was aimed of isolation and characterization of onion associated spoilage bacteria from spoiled onions. The experimental study design was used and the study was conducted at Bahir Dar University (BDU), Microbiology laboratory from February to May, 2018. The onion sample were purchased, put in to sterile polyethylene bag and transported in to BDU, microbiology laboratory and processed after 3 hours. Enumeration of total viable count and isolation of spoilage bacteria, morphological and biochemical characterization of the obtained bacteria were done to isolate and characterize onion associated bacteria. Confirmatory test was carried out to test whether the isolated bacteria are spoilage causative agent or not by re inoculating the bacteria in to healthy onion samples. The mean total viable count was log 8.1CFU/ml. A total of nine bacteria were recovered from spoiled onion samples and identified as Bacillus spp. (8.6%), Staphylococcus spp. (9.5%), two Serratia spp, (11.9%), Pseudomonas spp. (21.9%), Enterobacter spp. (16.7%), Erwinia spp. (13.3%), Enterococcus spp. (6.2%) and Micrococcus spp. (11.9%). All of the isolated bacteria were confirmed as onion spoilage causative agent. In general it can be concluded that onion can harbor different spoilage causing bacteria and also can be conclude that gram negative bacteria are higher in count than gram positive bacteria in spoiled onion. Keywords: Onion (Allium cepa), spoilage bacteria, postharvest loss