This study investigated the effect of macrospora leaf spot (MLS), caused by Stenocarpella macrospora, on photosynthetic gas exchange parameters and chlorophyll a fluorescence parameters determined in leaves of plants from two maize cultivars ('ECVSCS155' and 'HIB 32R48H') susceptible and highly susceptible, respectively, to S. macrospora. MLS severity was significantly lower in the leaves of plants from ECVSCS155 relative to the leaves of plants from HIB 32R48H. In both cultivars, net CO2 assimilation rate, stomatal conductance, and transpiration rate significantly decreased, while the internal to ambient CO2 concentration ratio increased in inoculated plants relative to noninoculated plants. The initial fluorescence and nonphotochemical quenching significantly increased in inoculated plants of ECVSCS155 and HIB 32R48H, respectively, relative to noninoculated plants. The maximum fluorescence, maximum PSII quantum efficiency, coefficient for photochemical quenching, and electron transport rate significantly decreased in inoculated plants relative to noninoculated plants. For both cultivars, concentrations of total chlorophyll (Chl) (a+b) and carotenoids and the Chl a/b ratio significantly decreased in inoculated plants relative to noninoculated plants. In conclusion, the results from the present study demonstrate, for the first time, that photosynthesis in the leaves of maize plants is dramatically affected during the infection process of S. macrospora, and impacts are primarily associated with limitations of a diffusive and biochemical nature.