Osteosarcoma is a highly aggressive tumor that originates in the bone and often infiltrates nearby bone cells. It is the most prevalent type of primary bone cancer among the various bone malignancies. Traditional cancer treatment methods such as surgery, chemotherapy, immunotherapy, and radiotherapy have had restricted success. However, the integration of nanotechnology into cancer research has led to notable progress. One promising area is the use of marine-derived polysaccharide-based nano formulations for treating various human diseases, including cancer. This study presents a straightforward method for synthesizing biocompatible gold nanoparticles (AuNPs), utilizing sodium borohydride as a reducing agent and a cost-effective, water-soluble chondroitin sulfate (CS) derived from shark cartilage as a stabilizing agent. The synthesized CS-Au NPs appeared purple and were mainly spherical, with 40.768 nm of average size. Cytotoxicity assays (MTT) indicated that CS-Au NPs significantly reduced the viability of human osteosarcoma cells (MG63) at 100 μg/mL, while it showed no cytotoxic effects on mouse embryonic fibroblast cells (NIH3T3) at the same concentration. The observed toxicity of the CS-Au NPs was linked to a rise in the production of reactive oxygen species (ROS) within damaged mitochondria. ROS generation and changes in mitochondrial membrane potential were detected in MG63 cells treated with CS-Au NPs. Furthermore, apoptotic analysis through ethidium bromide dual staining and flow cytometry demonstrated that CS-Au NPs at higher concentrations significantly increased the amount of apoptotic cells, as demonstrated by acridine orange/ethidium bromide staining. Flow cytometry also confirmed that CS-Au NPs activated the apoptotic pathway in MG63 cells.