Haemophilus influenzae and Streptococcus pneumoniae are commensal bacteria that inhabit the human nasopharynx. Both species have the capacity to migrate from this anatomical niche and in doing so cause various diseases. Previous studies have shown that both H. influenzae and S. pneumoniae are present in middle ear tissues excised from chronic Otitis Media patients. However, although both species do co-exist within a biofilm, the study of the interplay between these pathogens is relatively recent, and there remains controversy about the interactions observed between these pathogens. This study investigates the interactions that occur between H. influenzae and S. pneumoniae in both the planktonic and biofilm state in co-culture conditions. To date, this study has determined that S. pneumoniae predominated over H. influenzae in both the planktonic and biofilm state in co-culture, dramatically reducing H. influenzae viability. This effect did not appear to be strain specific, and held true for the laboratory and clinical isolate strains of both species. This competitive interaction was observed to occur in co-culture after 24hrs, but not after 6hrs, suggesting a growth-phase specific competition. In addition, the effect of pH was shown to be important in these competitive interactions; competition was not observed at an alkaline pH, but did occur at neutral or acidic pH. The competitive effect was shown to not be mediated by soluble secreted factors of S. pneumoniae. Transcriptomic analysis by mRNA sequencing showed that S. pneumoniae modified H. influenzae gene expression, in particular resulting in an upregulation of stress response genes, membrane associated proteins and the holin and endolysin genes of the φFlu bacteriophage. The results suggest that there are complex molecular interactions between H. influenzae and S. pneumoniae and these are significant in the colonization by both pathogens within the one site in the human host.