There is growing interest in the use of magnetic resonance spectroscopy (MRS) to relate brain functions with the concentrations of glutamate (Glu; chief excitatory neurotransmitter) and gamma-aminobutyric acid (GABA; chief inhibitory neurotransmitter) in specific regions. However, it remains unclear how robustly MRS-measured Glu and GABA reflect activities of excitatory and inhibitory neurons, respectively. To address this issue, we conducted MRS measurements of Glu and GABA along with large field-of-view, two-photon mesoscopic imaging of calcium signals in excitatory and inhibitory neurons in the same brain region of living, unanesthetized mice. For monitoring stimulus-driven activations of a brain region, MRS signals and mesoscopic neural activities were measured during two consecutive sessions of 15-min prolonged sensory stimulations. In the first session, activities of putative excitatory neurons were increased, while activities of inhibitory neurons remained at the baseline level. In the second half, while activities of excitatory neurons remained elevated, activities of inhibitory neurons were significantly enhanced. Correspondingly, the concentration of Glu increased without changes in GABA levels in the first MRS session, and the GABA concentration markedly increased from the baseline in the second session. We also assessed regional neurochemical and functional statuses related to spontaneous neural firing by measuring MRS signals and neuronal activities in a mouse model of Dravet syndrome under a resting condition without any task and stimuli. Mesoscopic assessments showed that activities of inhibitory neurons in the cortex were diminished relative to wild-type mice in contrast to spared activities of excitatory neurons. Consistent with these observations, the Dravet model exhibited lower concentrations of GABA than wild-type controls. Collectively, the current investigations demonstrate that the MRS-measured Glu and GABA can reflect spontaneous and stimulated activities of neurons producing and releasing these neurotransmitters in an awake condition.