Aim Metformin is a widely used antidiabetic drug associated with the rare side effect of lactic acidosis which has been proposed to be linked to drug‐induced mitochondrial dysfunction. Using respirometry, the aim of this study was to evaluate mitochondrial toxicity of metformin to human blood cells in relation to that of phenformin, a biguanide analogue withdrawn in most countries due to a high incidence of lactic acidosis. Methods Peripheral blood mononuclear cells and platelets were isolated from healthy volunteers, and integrated mitochondrial function was studied in permeabilized and intact cells using high‐resolution respirometry. A wide concentration range of metformin (0.1–100 mm) and phenformin (25–500 μm) was investigated for dose‐ and time‐dependent effects on respiratory capacities, lactate production and pH. Results Metformin induced respiratory inhibition at complex I in peripheral blood mononuclear cells and platelets (IC50 0.45 mm and 1.2 mm respectively). Phenformin was about 20‐fold more potent in complex I inhibition of platelets than metformin. Metformin further demonstrated a dose‐ and time‐dependent respiratory inhibition and augmented lactate release at a concentration of 1 mm and higher. Conclusion Respirometry of human peripheral blood cells readily detected respiratory inhibition by metformin and phenformin specific to complex I, providing a suitable model for probing drug toxicity. Lactate production was increased at concentrations relevant for clinical metformin intoxication, indicating mitochondrial inhibition as a direct causative pathophysiological mechanism. Relative to clinical dosing, phenformin displayed a more potent respiratory inhibition than metformin, possibly explaining the higher incidence of lactic acidosis in phenformin‐treated patients.