This study explores rutin (RT), rutin nanoparticles (RTN), and hyaluronic acid-coated rutin nanoparticles (HA RTN) effects on essential cellular processes in MCF-7 breast cancer cells. Analyzing cell cycle dynamics, apoptosis induction, intracellular reactive oxygen species (ROS) levels, and cellular uptake aimed to understand their influence on cancer cell behaviour. Treatment induced changes in cell cycle distribution, reducing cells in the G2 phase, suggesting a possible G2 arrest and associated cytotoxicity aligned with apoptosis pathways. Assessment of apoptosis revealed a significant increase across all treatments. Notably, HA RTN showed the most substantial increase in apoptotic cells, implying its potential for programmed cell death induction. Concurrently, ROS level evaluation highlighted notable increases, especially in HARTN-treated cells, indicating enhanced rutin accumulation and ROS production. Cellular uptake analysis demonstrated robust drug internalization, particularly in HA RTN-treated cells, indicating its efficient cellular penetration compared to RT and RTN formulations. In conclusion, HA RTN exhibited superior cytotoxicity, prominently induced apoptosis, elevated intracellular ROS levels significantly, and notably enhanced drug internalization in MCF-7 cells. These findings position HA RTN as a promising avenue for targeted cancer therapy and suggest its potential role in innovative treatment strategies.