Respiratory pathogens are transmitted through exhaled droplets and aerosols containing infectious respiratory particles (IRPs), where their persistence is shaped by rapid physicochemical transformations during drying. Although inactivation varies with microbial species, respiratory matrix composition, and environmental conditions, the underlying mechanisms remain poorly resolved. This study aims to quantify inactivation of surrogate bacterial pathogens, identify the dominant environmental and chemical drivers, and develop predictive relationships applicable to indoor air conditions. To avoid high-level biosafety requirements, two bacterial surrogates were selected: Escherichia coli (gram-negative) and Staphylococcus epidermidis (gram-positive). Experiments were performed with 1 µL artificial saliva droplets equilibrated at controlled relative humidities (30%, 50%, 70%) and constant temperature. Inactivation was quantified using colony enumeration and digital PCR. Droplet evaporation behavior was characterized, and efflorescence time was determined by filming the drying droplets. The Respiratory Aerosol Model (ReSAM) was used to simulate solute concentration dynamics and calculate changes in osmotic pressure and ionic strength during drying. For both E. coli and S. epidermidis, inactivation strongly correlated with increases in osmotic pressure (ΔΠ) at the point of droplet efflorescence, with the effect intensifying at lower relative humidity. Numerical fits relating inactivation to ΔΠ enabled prediction across additional RH conditions and different respiratory matrices. These findings highlight distinct inactivation patterns between gram-negative and gram-positive bacteria and demonstrate the central role of droplet chemistry in determining airborne microbial persistence. The results contribute to improved assessment of transmission risks and the development of strategies to reduce exposure in indoor environments.