This work highlights the critical role of synthesis conditions in tuning the properties of carbon dots (CDs) for optimized performance in biomedical applications, offering valuable insights into the design of these carbon nanomaterials. Although various synthesis methods and carbon sources have been explored for CD production, few studies have investigated how synthesis temperature modulates and optimizes their physicochemical attributes. In this study, cationic CDs derived from poly(ethylene imine) (PEI) and chitosan (CS) were synthesized using a microwave-assisted hydrothermal method at different temperatures to explore this aspect. It was found that higher carbonization temperatures during the hydrothermal process resulted in smaller, more photoluminescent CDs. This increase in temperature significantly enhanced the biological interactions of the CDs, demonstrating notable biocompatibility. In contrast, the lowest hydrothermal temperature enhanced cytotoxic effects against the Gram-positive pathogen under light exposure. Furthermore, gastric cancer (AGS), colon cancer (HT-29), cervical cancer (HeLa), prostate cancer (PC-3), and breast epithelial (MCF-10) cell lines showed cytotoxicity that was dependent on the CDs synthesized at different temperatures.