Methicillin-resistant Staphylococcus aureus (MRSA) infections are a global health burden with an urgent need for antimicrobial agents. Studies have shown that host immune responses limit essential metals such as zinc during infection, leading to the limitation of bacterial virulence. Thus, the deprivation of zinc as an important co-factor for the activity of many S. aureus enzymes can be a potential antimicrobial approach. However, the effect of zinc deprivation on S. aureus and MRSA is not fully understood. Therefore, the current study aimed to dissect the effects of zinc deprivation on S. aureus hemolytic activity and biofilm formation through employing biochemical and genetic approaches to study the effect of zinc deprivation on S. aureus growth and virulence. Chemically defined media (CDM) with and without ZnCl2, was used to assess the effect of zinc deprivation on growth, biofilm formation, and hemolytic activity in methicillin-susceptible S. aureus (MSSA) RN6390 and MRSA N315 strains. Zinc deprivation decreased the growth of RN6390 and N315 S. aureus strains significantly by 1.5–2 folds, respectively compared to the zinc physiological range encountered by the bacteria in the human body (7–20 µM) (p < 0.05). Zinc deprivation significantly reduced biofilm formation by 1.5 folds compared to physiological levels (p < 0.05). Moreover, the hemolytic activity of RN6390 and N315 S. aureus strains was significantly decreased by 20 and 30 percent, respectively compared to physiological zinc levels (p < 0.05). Expression of biofilm-associated transcripts levels at late stage of biofilm formation (20 h) murein hydrolase activator A (cidA) and cidB were downregulated by 3 and 5 folds, respectively (p < 0.05) suggested an effect on extracellular DNA production. Expression of hemolysins-associated genes (hld, hlb, hla) was downregulated by 3, 5, and 10 folds, respectively, in absence of zinc (p < 0.001). Collectively the current study showed that zinc deprivation in vitro affected growth, biofilm formation, and hemolytic activity of S. aureus. Our in vitro findings suggested that zinc deprivation can be a potential supportive anti-biofilm formation and antihemolytic approach to contain MRSA topical infections.