Over the last decade, it has become clear that the pollution by plastic debris presents global societal, environmental, and human health challenges. Increasing scientific attention on the occurrence and influence of plastic particles, and their potential toxic effects on the environment are evident. However, humans are also exposed to plastics in daily life and very limited information is available concerning human health, especially aging process in biological fluids and the colonization of the bacteria on plastics due to transport the pathogens, causing the infectious is another vital problem. Moreover, various studies also observed the importance of the nano-sized particles since a lower size can increase the activity, surface area etc. Therefore, the aim of this study is to investigate the in vitro (artificial saliva, artificial lysosomal fluid, phagolysosomal simulant fluid, Gamble’s solution) aging of plastic particles and the effect on the formation of Staphylococcus aureus biofilms with nanoplastics under in vitro conditions. We found that the surface properties of the nano-sized plastic particles changed according to the surrounding environment and surface deformation and the surface affinity to the biological activity was triggered with in vitro conditions due to higher hydroxyl index and carbon to nitrogen ratio. The results also showed that the Staphylococcus aureus biofilms formed with nanoplastics with the aging in vitro conditions. However, their levels and characteristic of extracellular polymeric substances were varied in these conditions with aging compared to control, and more basophilic extracellular polymeric substances were formed with these conditions.