Low-level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects induced by LLLT presumably scale from photobiostimulation/photobioinhibition at the cellular level to the molecular level. The detailed mechanism underlying this effect remains unknown. This study quantifies some relevant aspects of LLLT related to molecular and cellular variations. Malignant breast cells (MCF-7) were exposed to spatially filtered light from a He-Ne laser (633 nm) with fluences of 5, 28.8, and 1000 mJ/cm2. The cell viability was evaluated by optical microscopy using the Trypan Blue viability test. The micro-Fourier transform infrared technique was employed to obtain the vibrational spectra of each experimental group (control and irradiated) and identify the relevant biochemical alterations that occurred due to the process. It was observed that the red light influenced the RNA, phosphate, and serine/threonine/tyrosine bands. We found that light can influence cell metabolism depending on the laser fluence. For 5 mJ/cm2, MCF-7 cells suffer bioinhibition with decreased metabolic rates. In contrast, for the 1 J/cm2 laser fluence, cells present biostimulation accompanied by a metabolic rate elevation. Surprisingly, at the intermediate fluence, 28.8 mJ/cm2, the metabolic rate is increased despite the absence of proliferative results. The data were interpreted within the retrograde signaling pathway mechanism activated with light irradiation.