Photobiomodulation (PBM), an emerging and non-invasive intervention, has been shown to benefit the nervous system by modifying the mitochondrial cytochrome c-oxidase (CCO) enzyme, which has red (620–680 nm) or infrared (760–825 nm) spectral absorption peaks. The effect of a single 810-nm wavelength with a combination of 810 nm and 660 nm lights in the brain metabolic activity of male and female rats was compared. PBM, with a wavelength of 810 nm and a combination of 810 nm and 660 nm, was applied for 5 days on the prefrontal cortex. Then, brain metabolic activity in the prefrontal area, hippocampus, retrosplenial, and parietal cortex was explored. Sex differences were found in cortical and subcortical regions, indicating higher male brain oxidative metabolism, regardless of treatment. CCO activity in the cingulate and prelimbic area, dentate gyrus, retrosplenial and parietal cortex was enhanced in both treatments (810 + 660 nm and 810 nm). Moreover, using the combination of waves, CCO increased in the infralimbic area, and in CA1 and CA3 of the hippocampus. Thus, employment of a single NIR treatment or a combination of red to NIR treatment led to slight differences in CCO activity across the limbic system, suggesting that a combination of lights of the spectrum may be relevant.