This study investigates synthesizing and characterizing dual-doped carbon dots (N– CD) using malic acid, oxalic acid, diammonium tartrate, and rare earth metal salts as precursors. The research highlights the impact of varying dopant ratios on the N– CD structural, morphological, optical properties, and antimicrobial activity. Results demonstrate that adjusting dopant ratios significantly influences photoluminescence behavior. Notably, malic acid-oxalic acid co-doped CD exhibit a red-shift in emission from 443 nm to 477 nm as oxalic acid content increases from 3 g to 5 g, attributed to additional surface defects and carboxyl groups. Conversely, the diammonium tartrate-oxalic acid system shows a slight blue-shift from 478 nm to 475 nm due to sp³ defects that widen the bandgap. Emission intensity is notably higher for the diammonium tartrate-oxalic acid system, ranging from 30,620 to 66,260 counts, compared to the malic acid-oxalic acid system (6,260 to 42,708 counts), underscoring nitrogen doping’s role in enhancing luminescence, incorporating rare earth metals like terbium and europium results in crystalline TbN and EuN phases, improving photoluminescent properties and new radiative pathways. The N– CD demonstrate significant antimicrobial activity under UV radiation, with terbium and europium co-doped N– CD showing enhanced microbial inactivation compared to undoped N– CD. The antimicrobial efficacy was attributed to the enhanced generation of reactive oxygen species (ROS) under UV light, effectively inactivating bacterial strains. These findings indicate that the dual-doped N– CD possess tunable optical properties and enhanced antimicrobial activity, making them suitable candidates for applications in bioimaging, sensing, and disinfection technologies.