Rising antibiotic resistance necessitates alternatives such as silver nanoparticles (AgNPs), which exhibit bactericidal activity via multi-target mechanisms (e.g., membrane disruption, ROS production). While resistance to chemically synthesized AgNPs exists, the potential for resistance to green-synthesized AgNPs, such as those from reishi mushroom, is unknown. This study compared S. aureus resistance development against both AgNP types using experimental evolution by analyzing genomic and morphological changes. Additionally, this work evaluated potential cross-resistance responses to ionic silver and investigated how adaptation to green-synthesized AgNPs affects sensitivity to chemically synthesized AgNPs (and vice versa). Rapid resistance, along with cross-resistance to silver ions, emerged in bacteria following 14 days of sublethal exposure to silver nanoparticles, regardless of whether they were chemically or biologically synthesized. While green-synthesized AgNPs demonstrated a substantial resistance to chemical variants (p < 0.05), the reverse effect was not as strong, and resistant populations showed distinct morphological adaptations. Genomic analysis highlighted convergent hard selective sweeps, identifying common mutations across both chemical and green AgNP-treated populations, with limited unique mutations found for either. These findings enhance our understanding of bacterial resistance mechanisms to nanomaterials, contributing to the development of safer, eco-friendly, and high-efficacy treatments against multidrug-resistant infection.