Agriculture has the most significant contribution in fulfilling the basic human need, sustaining life, and strengthening the economy of any country. To feed the exploding population of the world, there has been a quantum jump in the production of agricultural commodities, which has led to the production of a substantial considerable quantity of agricultural and agro-industrial wastes. The bulks of these wastes are lignocellulosic in nature and consist of three main polymeric constituents, i.e., cellulose, hemicellulose, and lignin, which are recalcitrant. The primary significant portions of these remain unutilized and are burnt in the field, leading to severe environmental aggression and wastage of resource. Farmers across the globe, including India, burn these agricultural wastes in their thousands of acre land, which contribute to spoiling the air quality index (AQI). This is very harmful, especially to children, pregnant women, old adults, and for patients suffering from respiratory diseases. The current manuscript sets up an agro-waste management platform by using paddy straw as a substrate for the production of nutritionally and medically rich oyster mushroom, Pleurotus florida (Pf) and which is further used in the green synthesis of bimetallic (gold-platinum) Au-Pt nanoparticle. Yield performance and biological efficiency of Pf were calculated from the degraded paddy straw. The green synthesized Au-Pt NPs were structurally characterized by ultraviolet-visible (UV-Vis), X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), and zeta potential analysis. The prepared NPs showed a face-centered cubic crystal structure, icosahedral shape with a mean particle size of 16 nm. Furthermore, we examined the cytotoxic activity of Au-Pt NPs using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, intracellular reactive oxygen species (ROS) generation, and apoptosis by propidium iodide assay. We found that Au-Pt NPs exerted apoptotic activity on the human colon cancer cell line (HCT 116) in a dose-dependent manner from 12.5 to 200 μg/mL. Overall, our findings create a prototype and open a new door to synthesizing functional nanoparticle by using oyster mushroom as the substrate for paddy straw agro-waste management and the applicability of Pf in the synthesis of eco-friendly Au-Pt NPs. This is the first kind of approach that kills two birds with one stone.