This study explores defect engineering in 2D materials using ion beam irradiation to modify the electrical and optical properties with potential in advancing quantum electronics and photonics. Helium and neon ions ranging from 5 to 7.5 keV are employed to manipulate charge transport in monolayer molybdenum disulfide (MoS₂). In situ electrical characterization occurs without vacuum breakage post-irradiation. Raman and photoluminescence spectroscopy quantify ion irradiation's impact on MoS₂. Small doses of helium ion irradiation enhance monolayer MoS₂ conductivity in field-effect transistor geometry by inducing doping and substrate charging. Findings reveal a strong correlation between the electrical properties of MoS₂ and the primary ion used, as well as the substrate on which the irradiation occurred. Using hexagonal boron nitride (h-BN) as a buffer layer between MoS₂ flake and SiO₂ substrate yields distinct alterations in electrical behavior subsequent to ion irradiation compared to the MoS₂ layer directly interfacing with SiO₂. Molecular dynamics simulations and density functional theory provide insight into experimental results, emphasizing substrate influence on measured electrical properties post-ion irradiation.