Shiga-toxigenic encoding Escherichia coli are a global health concern. Carriage of the shigatoxin gene increases the pathogenicity of the bacteria as the toxin has downstream impact on clinical disease. Enterohaemorrhagic E. coli (EHEC) symptoms lead from mild to severe bloody diarrhoea, where the toxin targets protein synthesis in specific cells leading to cell death and clinical sequalae including; haemolytic ureamic syndrome (HUS), haemorrhagic colitis (HC) and thrombotic thrombocytopenic purpura (TTP). This toxin is carried by lambdoid-like bacteriophages. Temperate bacteriophages play an underestimated role in microbial evolution, especially microbial infection and disease progression. Using comparative metabolite profiling and targeted GC-MS of fatty acid methyl esters we demonstrate the impact of the Shiga toxin-prophage ɸ24B on its Escherichia coli host. We show that as a lysogen, the prophage alters the physiology of its host cell by decreasing the total fatty acid composition, either through catabolism or down regulation. Our previous work showed phage-mediated control of the biotin pathway and it being rate limiting to growth. The biotin pathway is intrinsically linked to the fatty acid synthesis pathway and is likely manipulated by the same phage mechanisms. Intriguingly distinct strategies in host cell wall fatty acids are noticed when treated with antimicrobials 8-hydroxyquinoline or chloroxylenol. When stressed, alongside increasing the total cell wall lipids compared to the naïve host, there is a significant increase in Cyclopropaneoctanoic acid, 2-octyl- and dodecanoic acid. We hypothesise that when the host is not challenged by the antimicrobial the phage manipulates the fatty acid synthesis pathway to redirect energy and resources from cell wall physiology. We further hypothesise under antimicrobial challenge phage infection promotes broad antimicrobial resistance by increasing total cell wall lipids, significantly increasing fatty acids that alter membrane fluidity.