Two-dimensional infrared spectroscopy (2DIR) was applied to phylloquinone (PhQ), an important biological cofactor, to elucidate the impact of hydrogen bonding on the ultrafast dynamics and energetics of the carbonyl stretching modes. 2DIR measurements were performed on PhQ dissolved in hexanol, which served as the hydrogen bonding solvent, and hexane, which served as a non-hydrogen bonding control. Molecular dynamics simulations and quantum chemical calculations were performed to aid in spectral assignment and interpretation. From the position of the peaks in the 2DIR spectra, we extracted the transition frequencies for the fundamental, overtone, and combination bands of hydrogen bonded and non-hydrogen bonded carbonyl groups of PhQ in the 1635–1680 cm–1 region. We find that hydrogen bonding to a single carbonyl group acts to decouple the two carbonyl units of PhQ. Through analysis of the time-resolved 2DIR data, we find that hydrogen bonding leads to faster vibrational relaxation as well as an increase in the inhomogeneous broadening of the carbonyl groups. Overall, this work demonstrates how hydrogen bonding to the carbonyl groups of PhQ presents in the 2DIR spectra, laying the groundwork to use PhQ as a 2DIR probe to characterize the ultrafast fluctuations in the local environment of natural photosynthetic complexes.