This study explores the dose-dependent effects of 660-nm and 808-nm photobiomodulation (PBM) on mitochondrial oxygen respiration rate activity in MG-63 osteoblast cells using an innovative 3D in vitro spheroid model. MG-63 osteoblast cells were grown to 80% confluence and seeded in fish gelatin hydrogel (LunaGel™) to form 3D spheroids within 3–7 days. Spheroids were seeded on Seahorse microplates and incubated in a LunacrossLinker™ (visible light crosslinking system) for 2 min to give hydrogel a mid-stiffness of 3.5 kPa. Cells were exposed to PBM either 660-nm or 808-nm at panel setting of 5 J/cm2 and 15 J/cm2 and then assessed immediate (15 min before analysing) and 24 h time points. Mitochondrial activity was determined using an XFe96 Seahorse analyzer. Data distribution was assessed, and parametric or non-parametric tests and compared the mitochondrial respiratory capacity across different experimental conditions. The study indicated that 660-nm and 808-nm PBM could modulate mitochondrial functions in osteoblasts. The maximal respiratory rate for the fluency assessed at 808-nm wavelength was increased when cells were assessed immediate post. Interestingly, the 660-nm PBM-treated cells showed a decrease in oxygen consumption rate (OCR) at the basal and maximal bioenergetic state at all time points (immediate and 24 h.) and fluency compared to the untreated control. The effects of 660-nm and 808-nm wavelengths on osteoblast mitochondrial function suggest that PBM demonstrates differential modulation of osteoblast metabolism and bioenergetics depending on the wavelength. These findings have practical implications in both research and clinical settings, offering insights into selecting specific wavelengths for therapeutic applications.