The electrocatalytic activity towards formic acid oxidation reaction (FAOR) in the presence of simultaneous oxygen reduction reaction (ORR) displayed by 5 different metallic nanoparticles (NPs) (Pt100, Pt75Pd25, Pt50Pd50, Pt25Pd75 and Pd100) was studied and compared using chronoamperometry and the micropipette delivery/substrate collection (MD/SC) mode of the scanning electrochemical microscopy (SECM). This is of special interest for understanding the O2 crossover effect in direct formic acid fuel cells (DFAFCs) and to search highly selective electrocatalysts useful in mixed-reactant fuel cells (MRFCs). A detailed analysis of the SECMresults in comparison with chronoamperometry demonstrates, for the first time, the relevant role played by dissolved O2 in solution on the Pd100 NPs deactivation during FAOR, which cannot be explained neither by the specific adsorption of dichloroethane (DCE) on Pd nor by a simple addition of two opposed currents coming from simultaneous FAOR and ORR. Two main mechanistic factors are proposed for explaining the different sensitivity towards O2 presence in solution during FAOR when comparing Pd- and Pt-rich catalysts. On the one hand, the relevance of H2O2 production (ORR byproduct) and accumulation on Pd NPs,which alters its performance towards FAOR. On the other hand, the predominance of the poisoning pathway forming COads during FAOR on Pt NPs, whose oxidation is facilitated in the presence of traces of O2. Interestingly, the deactivation effect displayed on Pd100 NPs during FAOR due to the H2O2 generation and accumulation becomes negligible if a convective regime is applied in solution. SECMis proved as a fast and powerful technique for studying O2 crossover effect in different electrocatalysts and for identifying highly selective electrocatalysts candidates forMRFCs. In particular, among the samples evaluated, Pt75Pd25 NPs present the highest average performance for FAOR in 0.5 M H2SO4 solution in the presence of O2 within the potential range under study (0.3–0.7 V vs RHE).