The accumulation of agricultural and industrial residues inevitably contributes to environmental pollution. Thus, several scientific investigations have been conducted to overcome this problem and to add an economic value proposition. Unlike typical sugarcane bagasse applications, this work presents a novel application of sugarcane bagasse waste in a green synthesis approach for forming zinc oxide nanoparticles (ZnO-NPs). This work opens the door to studying the potential of sugarcane bagasse in a green synthesis orientation. Phytochemical assessment of the aqueous extract of sugarcane bagasse waste was conducted by studying total flavonoid content, total phenolic content, and antioxidant assays. ZnO-NPs were synthesized using the aqueous sugarcane bagasse extract (ASCBE) with a 96% yield. To obtain 99.7% pure ZnO-NPs, nanoparticles were calcined at 550 °C to remove any remaining plant extract residues. The purity and yield of the produced and modified ZnO-NPs were studied. The initially produced and modified ZnO-NPs were characterized using XRD, FT-IR, UV, TEM, TGA, and PL and to determine the necessity of the calcination step. A detailed proposed mechanism for the formation of ZnO-NPs mediated by ASCBE was introduced. The ZnO-NPs were studied for their antibacterial, antifungal, and antiviral activities. The ZnO-NPs before calcination were found to exhibit more potent antimicrobial activity against both P. aeruginosa and A. niger compared to the calcined ZnO-NPs. In addition, molecular docking analysis revealed that the ZnO-NPs had the strongest binding affinity towards the P. aeruginosa RhlG/NADP active-site complex and the crystal structure of Actibind, a T2 RNase of A. Niger. ZnO-NPs also showed promising binding interactions with viral targets, including the Herpes simplex virus type II protease and Influenza virus NS1 effector domain. Additionally, environmental and economic studies were achieved to relate the scientific study with daily life applications.