Bacterial cellulose (BC) stands out as a prominent biopolymer of global importance, distinguished by its unique advantages over plant-derived cellulose. Strains such as Acetobacter xylinum, renowned for their proficient BC production, draw considerable attention in both commercial and biomedical areas. This research aimed to selectively isolate cellulose-producing bacteria with enhanced efficiency from a variety of fruit samples utilizing a cost-effective methodology. A total of 60 fruit samples were selected, and the assessment focused on 17 strains derived from rotten banana, red apple, green apple, and pineapple samples. The evaluation encompassed an examination of bacteriological traits and cellulose synthesis, with subsequent identification of strains achieved through DNA extraction and 16S rRNA PCR analysis. The experimental findings reveal cellulose-producing strains, including model A. xylinum (KCCM 40407) obtained from the Pharmacy lab of COMSATS University Islamabad Abbottabad Campus, Pakistan, designated as number 2, serving as a control. Notably, strains isolated from deteriorated fruits (samples 1, 4, 8, 11, 12, and 15) demonstrated the capacity to produce soluble cellulose. A. xylinum (model strain 2) was cultured under static conditions in HS media, demonstrating remarkable efficacy for cellulose sheet production. Subsequent characterization employing scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) unveiled a nano-fiber mat featuring multi-layered fibers. This eco-friendly approach has the potential for large-scale, high-quality cellulose production, applicable in biomedical and industrial fields. The research highlights an environmentally sustainable and economically viable method for cellulose production, presenting potential applicability across biomedical and industrial arenas on a significant scale.