Anaerobic co-digestion (AcoD) has been practiced for decades to convert waste into value-added energy products, especially biogas. This study aimed to assess the potential of biogenic methane (CH4) production from the co-digestion of pretreated cotton gin trash (CGT), cow manure, and sludge. CGT contains high cellulosic content, making it a reliable feedstock for biogenic methane production. To further improve the biogas quantity and quality, the CGT was subjected to physical pretreatments, i.e., hot water (HW), ultra-sonication (US), and a combination of both (HW+US). After 91 days of AcoD, 79–110 L of biogas was produced by the treatments. Among the treatments, HW+US-pretreated CGT presented maximum biogas production capacity, at 110 L. Besides, this treatment showed the high-quality biogenic CH4 content, 52.4% of the total biogas volume, with an improved conversion rate of 0.37 L/g of volatile suspended solids consumed. In addition, this study discussed the structural changes in feedstock due to pretreatments and correlated them with the corresponding biogenic methane production. The study reports the potential of pretreated CGT conversion to CH4. It will impact the circular economy by contributing to on-farm energy requirements and reducing the financial expenditures incurred in this regard.