The objective of this work was to evaluate the influence of herbicides 2,4-D and dicamba on the physiological response of chlorophyll a in olive (Olea europaea) seedlings. The following eight rates of the 2,4-D (670 g a.e. ha-1) and dicamba (720 g a.e. ha-1) herbicides were applied: 0, 1.56, 3.13, 6.25, 12, 25, 50, and 100% of the recommended for burndown. The herbicides were applied at 80 cm above the seedlings using a CO2 pressurized back sprayer with a 150 L ha-1 spray volume. Plant gas exchange was measured using a portable infrared gas analyzer. Chlorophyll a fluorescence was evaluated using a portable modulated light fluorometer. The application of both herbicides caused a decrease in the process of CO2 assimilation in the plants, reducing net photosynthesis and stomatal conductance. The 2,4-D herbicide caused the most severe effects on the variables related to chlorophyll a fluorescence. Low values of maximum quantic yield were observed after the application of the herbicides, of which dicamba was the most harmful. Both herbicides damage the photosynthetic apparatus of olive seedlings.