Mitochondria are the metabolic hub of the cell. During bacterial and viral infections, mitochondrial function is compromised leading to unregulated immune responses of the hosts’ cells.  In this study, we investigated mitochondrial dysfunction in murine cells stimulated with bacterial (LPS) and viral (Poly(I:C)) ligands. We utilized label-free two-photon excitation fluorescence (TPEF) imaging of endogenous fluorophores, reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) levels. In addition to evaluating mitochondrial function utilizing a standard metric such as the optical redox ratio, we developed a new metric for the first time based on the FAD-TPEF intensity to quantify mitochondrial organization. Our results show that TPEF imaging is a robust label-free method to examine cellular mitochondrial activity in immune cells such as macrophages.