Following screening of 4 strains of Escherichia coli O157:H7 (E32511, E318N, H4420N, and R508N) for acid tolerance, strain H4420N was selected for further study into the influence of pH on bactericidal activity of 6 fatty acids (capric, lauric, palmitic, oleic, linoleic, and linolenic). Strain H4420N was cultured for 6 h in Luria–Bertani broth amended with individual fatty acids at 20 mmol/L, with pH adjusted to 7.0, 4.3, or 2.5. None of the fatty acids exhibited bactericidal activity at pH 7.0 (p >0.05). At pH 4.3, only capric, lauric, and linoleic acids reduced viability of H4420N (p < 0.05). At pH 2.5, oleic (C18:1) and linolenic (C18:3) acids had modest effects on H4420N viability, whereas capric (C10:0), lauric (C12:0), and linoleic (C18:2) acids resulted in a reduction ≥5 log10colony-forming units (CFU)/mL (p < 0.05). Capric and lauric acids were examined further at pH 2.5 over a range of concentrations (0.15–20 mmol/L). After 10 min of exposure, 5 log10 CFU/mL reductions (p < 0.05) were achieved by lauric acid at 2.5 mmol/L and by capric acid at 0.31 mmol/L. Acid stress increased the sensitivity of acid-tolerant E. coli O157:H7 strain H4420N to fatty acids. Including sources of these fatty acids in diets for cattle might impair the ability of this zoonotic pathogen to survive passage through the stomach, possibly reducing the potential for its colonization in the lower gut.