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Introduction: The ripening of fleshy fruits is a developmental process that involves changes in color, texture, aroma, nutrients, and diversity of microbiomes. Some microorganisms, specially, bacteria and molds are responsible for postharvest spoilage of fruits. Thus, this study is aimed at evaluating the alterations in microbiome and physico-chemical properties of selected fruits at different ripening stages., Methods: Totally, 108 fresh fruit samples of Musa paradisiaca, Citrus sinensis and Carica papaya at three ripening stages were collected and processed in this study. The biochemical methods and MALD-TOF MS were used in identification. The physico-chemical properties of all samples were analyzed using standard methods., Results: The minimum counts (6.74± 0.48-6.76± 0.42 log CFU/mL) and the maximum count (7.51± 0.43-7.96± 0.34 log CFU/mL) of AMB in all fruit samples was observed at mature green and overripe stages of the fruits, respectively. The ripening stage has significantly affected the microbial counts (P < 0.05) in all fruits, except counts of Enterobacteriaceae in banana and orange, and fungal counts in orange. The bacterial community of all fruits was predominated by B. cereus (33.7%), A. faecalis (17.3%), P. putida (15.2%), M. morganii (11.1%), S. sciuri (6.6%) and S. epidermidis (4.9%); while the fungal microbiome was constituted by Candida spp. (33.9%) followed by Saccharomyces spp. (18.1%) and Aspergillus spp. (16.3%). The ripening stages have also significantly affected the physico-chemical property in all samples. Accordingly, the lowest pH (3.53) and highest content of ascorbic acid (69.87 mg/100g) were observed in mature green oranges and overripe papaya, respectively, while the maximum concentration of total sugar (17.87%) and reducing sugar (14.20%) were recorded in overripe bananas., Conclusion: The presence of some potential human pathogens and spoilage microorganisms in fruit samples could contribute to post-harvest product losses besides the potential health risk associated with consumption of the tainted fruits. Hence, proper safety management practices and preservation mechanisms should be developed and put in place to ensure consumers safety against pathogens besides minimizing product losses through microbial spoilage., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kifle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)