Food losses have become a major issue worldwide. Approximately 1.3 billion tonnes of food are lost or wasted every year due to microorganisms, physical damage and oxidation of foods. As such, farmers of developing countries like Nepal, who also suffer from food losses of up to 45 % during post harvest face substantial economic burden. To overcome this problem, Guava leaves, which have been known to possess antioxidants and antibacterial properties, would be suitable for  local communities of Nepal to use as natural preservatives. Initially, food spoilage microorganisms were isolated from 7 different fruits and vegetables collected from Balkhu, Kalimati and Dhulikhel area, and identified through biochemical and molecular characterization. A total of  39 bacteria and 17 fungi were isolated, of which, the 16s rRNA and 18s rRNA regions of 34 bacteria and 10 fungi were partially sequenced and deposited at the National Centre for Biotechnology Information (NCBI) database. Secondly, Guava leaves were collected, washed and shade dried then crude extracts were extracted using different protocols (2 days, 25°C, 35°C, 100°C and 80°C). Antibacterial activity, phytochemical screening like total phenolics content, total flavonoid content, antioxidant tests were done and banana preservation efficacy under room conditions for all of the extracts. Among these, the extracts obtained from the 2 days protocol showed promising results against isolated food spoilage bacteria from bananas. Higher phytochemical properties were also observed from the same extract. Banana preservation study done under room conditions also favored the extract obtained from 2 days extraction protocol. Hence, it can be concluded that guava leaves could be a viable option for preparation of natural preservatives.