AuNPs have been extensively studied in various biomedical applications due to their unique physicochemical properties. Their properties are strongly dependent on their size and shape. Our previous work showed that rod-like AuNPs were able to affect the mitochondria function. In this present work, we investigated the shape effect of AuNPs on isolated rat liver mitochondria. Spherical and rod-like AuNPs were synthesized and incubated in a 10% BSA solution. The immediate effect of these particles on isolated mitochondria from Wistar rat liver (n = 4) was evaluated. Both morphologies reduced the mitochondrial O2 consumption, specifically in phosphorylative state (nmol/mg/mL): control 43.67 (± 9.68); spheres 26,69 (± 3.70); rods 26.30 (± 4.88); and maximal state (nmol/mg/mL): control 31.28 (± 5.69); spheres 7.20 (± 1.57); rods 8.93 (± 1.61), see Fig. C and D. They also decreased H2O2 production (Fig. E) and the mitochondrial swelling (Fig. F) in c.a. 30%. Under the conditions applied, spherical and rod-like AuNPs showed similar effects in the bioenergetic parameters evaluated, however, rod-like AuNPs were able to dissipate the mitochondrial electrochemical potential and spherical AuNPs were able to keep it, longer than the control (Fig. G). This particular behavior can be related to the shape or to the total surface area of the AuNPs present in the system. To solve this issue, we increased the number of spheres in the medium, reaching then the same surface area found on the rod-like particles system (Fig. I). Now, both systems showed similar effects in mitochondrial potential disruption. All these results shows that AuNPs act on mitochondria bioenergetic process, and particularly, the total surface area of AuNPs in the system is responsible to dissipate or keep the mitochondrial electrochemical potential.