This study investigates the impact of point defects on the electronic and optical properties of monolayer molybdenum disulfide (MoS₂). The research combines experimental techniques, such as aberration-corrected scanning transmission electron microscopy (ADF-STEM), with theoretical calculations using density functional theory (DFT). The defects, including sulfur vacancies and molybdenum antisites, are systematically identified and their concentrations determined. The study finds that the dominant defect category changes from sulfur vacancies in mechanically exfoliated and chemically vapor-deposited samples to molybdenum antisites in physically vapor-deposited samples. The influence of these defects on the electronic structure and charge-carrier mobility is predicted by DFT calculations and observed through electric transport measurements. The results highlight the importance of minimizing defects, especially antisites, for high-performance electronic devices, while controllably introducing them can potentially enhance magnetic properties. This comprehensive investigation advances the understanding of atomic defects in two-dimensional materials and paves the way for their application in optoelectronics and nanoelectronics.This study investigates the impact of point defects on the electronic and optical properties of monolayer molybdenum disulfide (MoS₂). The research combines experimental techniques, such as aberration-corrected scanning transmission electron microscopy (ADF-STEM), with theoretical calculations using density functional theory (DFT). The defects, including sulfur vacancies and molybdenum antisites, are systematically identified and their concentrations determined. The study finds that the dominant defect category changes from sulfur vacancies in mechanically exfoliated and chemically vapor-deposited samples to molybdenum antisites in physically vapor-deposited samples. The influence of these defects on the electronic structure and charge-carrier mobility is predicted by DFT calculations and observed through electric transport measurements. The results highlight the importance of minimizing defects, especially antisites, for high-performance electronic devices, while controllably introducing them can potentially enhance magnetic properties. This comprehensive investigation advances the understanding of atomic defects in two-dimensional materials and paves the way for their application in optoelectronics and nanoelectronics.