Coffee is a socioeconomic important plant all over the world due to its exportation and how it provides income to the farmers and the country. However, potato taste defect (PTD) affects the Rwandan coffee quality. The smell is reported to be caused by some bacteria that are responsible for the off‐flavor and may also be related to the infestation of Antestia pest which are in its elimination process. The aim of this study was to isolate, biochemically characterize, and identify bacteria producing potato flavor from Rwandan coffee. Five samples were obtained from different regions (Nyamasheke and Nyakizu) of Rwanda. Bacteria were isolated and enumerated in the nutrient agar media followed by culture on nutrient and tryptic soy broth media. Bacteria were also cultured to several carbon sources such as glucose, fructose, sucrose, starch, pectin, and galactose to smell the odor produced by those bacteria. DNA extraction of isolates was done, and the resulting DNA strands were undergone three steps of PCR to be amplified using the forward primer and reverse primer. The identification of bacteria producing potato flavor from Rwandan coffee beans was done through 16S rDNA method followed by sequence analysis using FinchTV software and BLAST. Earthy odor was the mostly produced one for nutrient agar and tryptic soy agar media, and for carbon sources such as sucrose, glucose, pectin, fructose, and galactose. The potato odor was recorded mostly from damaged floaters and hand‐sorted damaged coffee beans. However, other odors such as fruity and ferment were found to be produced by bacteria in coffee beans. The study came up by concluding the presence of different kinds of bacteria including Enterobacteriaceae and Pantoea, which are responsible for the formation of 2‐isopropyl‐3‐methoxypyrazine (IMP) in coffee beans and cause the production of potato flavor.