The persistence of intracellular Staphylococcus aureus [1] is an emerging paradigm through which to understand the high failure rate of gold-standard treatments for osteomyelitis associated with total hip and knee replacement surgery. As such, the characterisation of the interactions between these resident bacteria and osteocytes, the most abundant cell throughout the bone, is of primary importance for understanding not only mechanisms of treatment failure but also new modalities through which clinical cure of these recalcitrant infections can be achieved. An additional axis that needs consideration is how antibiotics influence this relationship as antibiotic treatments, especially at high doses and for extended durations are known to cause adverse effects upon host cells. Autophagy is a first-line innate defense mechanism against intracellular pathogens. In this study, the effects of modulation of autophagy on the clearance or persistence of intracellular S. aureus strain in a human osteocyte model [2] were studied. In addition, the effects of commonly used antibiotics at clinically relevant doses on this relationship were studied. Interestingly, whilst both promotion and blockade of autophagic flux significantly affected bacterial culturability from host cell lysates in both an acute and chronic infection context, neither of these treatments affected the absolute quantity of DNA from viable bacteria. Additionally, we demonstrated that treatment with vancomycin or rifampicin resulted in a modest but significant blockade of autophagic flux. Interestingly, these antibiotic treatments resulted in an increase in absolute DNA quantity from viable bacteria that was not consistent with the effects caused by autophagic modulation. In conclusion, whilst autophagic flux was demonstrated to have a significant impact on the growth phenotype of the resident bacteria, its modulation was not capable of affecting the number of viable bacteria. Additionally, whilst antibiotics were capable of blocking autophagy, the paradoxical increase in viable bacteria number suggests the involvement of additional host or bacterial mechanisms resulting in failure to clear S. aureus intracellular infection of osteocytes.