Streptococcus pneumoniae (pneumococcus) is a significant global pathogen responsible for more than 1 million deaths every year. To survive within the human host, this pathogen must evade both innate and adaptive immunity to proliferate and survive. As part of the innate immune response, the human host utilises long chain fatty acids, such as the polyunsaturated fatty acid, arachidonic acid (AA), in the defence against bacterial infection. However, the mechanisms by which AA contributes to bacterial clearance are not well understood. In this study, we applied a murine model of pneumococcal infection to investigate the contribution of AA to antimicrobial defence. Here, we show that the concentration of AA increased 44% in response to S. pneumoniae infection, suggesting a role in innate immune defence against the pneumococcus. The antimicrobial activity of AA was then analysed using THP-1 macrophages. This revealed that supplementation with AA increased pneumococcal killing by 50%, compared to un-supplemented macrophages. We then conducted RNA sequencing of S. pneumoniae in the presence of mild AA stress to determine the mechanism(s) of AA antimicrobial activity. These results show that AA stress caused ~4-fold down-regulation of the fatty acid biosynthesis gene cluster, with subsequent membrane composition analyses revealing a significant reduction in the abundance of endogenously produced fatty acids. Further, the abundance of AA in the membrane increased by 31%, leading to changes in membrane fluidity and integrity. Collectively, these data show that AA is selectively increased in host serum in response to pneumococcal infection. Increased AA abundance contributes to bacterial clearance through direct antimicrobial activity, predominately mediated though disruption of the pneumococcal membrane via concomitant down-regulation of endogenous fatty acid production and the physical insertion and accumulation of an exogenous fatty acid within the pneumococcal membrane.