Precise control of the optical and electrical properties of mono-layer (ML) thin MoS2 is crucial for future applications in functional devices. Depending on the synthesis route and the post-deposition annealing protocols, the number of sulfur vacancies in the material is different, which has a profound impact on the properties of the 2D layer. Here, we show that the sulfur vacancy-rich ML MoS2 films oxidize already at room temperature, which changes the photoluminescence (PL) yield, the MoS2–Al2O3 substrate interaction, and the structural integrity of the films. We used x-ray photoelectron spectroscopy to monitor the formation of MoO3 and possibly MoS3−xOx after exposure to air and to quantify the number of sulfur defects in the films. Atomic force microscopy measurements allow us to pinpoint the exact regions of oxidation and develop a dedicated low temperature heating procedure to remove oxidized species, leading to MoO3-free MoS2 films. AFM and Kelvin probe force microscopy show that the MoS2–Al2O3 substrate coupling is changed. The reduction in the MoS2–substrate coupling, combined with a preferential oxidation of sulfur vacancies, leads to a sevenfold increase in the PL intensity, and the ratio between trions and neutral excitons is changed. Our work highlights the importance of oxidized sulfur vacancies and provides useful methods to measure and manipulate their number in MoS2. Furthermore, changes in the MoS2–substrate interaction via sulfur vacancies and oxidation offer an elegant pathway to tune the optoelectronic properties of the two-dimensional films.