Biofilms are surface-attached communities of microorganisms surrounded by an extracellular matrix. The biofilm matrix plays an integral role in protecting the microorganisms within. Infections associated with biofilms are incredibly difficult to treat. Staphylococcus aureus is a major human pathogen and can produce robust biofilm. Candida parapsilosis is an emerging fungal pathogen that is frequently isolated as the cause of different types of infection. Its interaction with S. aureus has yet to be explored. The exploration of inter-species interaction between microorganisms presents an opportunity to gain insight into novel approaches to combat human pathogens by exploiting natural microbial antagonism. The aim of this project is to characterise the effect of co-culture with C. parapsilosis on S. aureus during biofilm formation. Using various in vitro techniques, such as a crystal violet biofilm assay, co-culture with C. parapsilosis resulted in an inhibition of robust S. aureus biofilm formation. Further investigation revealed that cell-free C. parapsilosis supernatant could also inhibit S. aureus biofilm formation. This result suggested that a fungal-secreted factor was causing the inhibitory effect on S. aureus biofilm. Extensive in vitro analysis of the bacterial biofilm inhibitory phenotype revealed that the fungal factor(s) exhibit pleiotropic effects and may be targeting various components of the S. aureus biofilm matrix. RNA sequencing identified global changes in transcription that occur in S. aureus in the presence of C. parapsilosis cells or cell-free supernatant. In addition, various in vitro techniques have been utilised to characterise the cell-free supernatant of C. parapsilosis, indicating that the active factors may be small molecules/metabolites. Future study is needed to identify the biofilm inhibiting factors produced by C. parapsilosis and assess their potential usefulness as an alternative or conjugate therapy against biofilm infections.