Finding a non-pathogenic surrogate aerosol that represents the deposition of typical bioaerosols in healthcare settings is beneficial from the perspective of hospital facility testing, general infection control and outbreak analysis. This study considers aerosolization of dilute aqueous lithium chloride (LiCl) and sodium chloride (NaCl) solutions as surrogate tracers capable of representing Staphylococcus aureus bioaerosol deposition on surfaces in mechanically ventilated rooms. Tests were conducted in a biological test chamber set up as a replica hospital single patient room. Petri dishes on surfaces were used to collect the Li, Na and S. aureus aerosols separately after release. Biological samples were analyzed using cultivation techniques on solid media, and flame atomic absorption spectroscopy was used to measure Li and Na atom concentrations. Spatial deposition distribution of Li tracer correlated well with S. aureus aerosols (96% of pairs within a 95% confidence interval). In the patient hospital room replica, results show that the most contaminated areas were on surfaces 2 m away from the source. This indicates that the room’s airflow patterns play a significant role in bioaerosol transport. NaCl proved not to be sensitive to spatial deposition patterns. LiCl as a surrogate tracer for bioaerosol deposition was most reliable as it was robust to outliers, sensitive to spatial heterogeneity and found to require less replicates than the S. aureus counterpart to be in good spatial agreement with biological results.