UV-A radiation is a tool that has a stimulant action in plants and can be used to induce benefits through the production of bioactive compounds and the synthesis of secondary metabolites, among others. Particularly, its application could stimulate tolerance to the biotic stress caused by Fusarium oxysporum f. sp. lycopersici (Fol) in tomato plants; for this reason, the aim of the present study was to evaluate the ability of UV-A radiation to stimulate tolerance against Fol. For this, a tomato crop was established in which two conditions of supplementation with UV-A radiation were tested on plants inoculated with the fungus Fol. The pathogen’s impact on the agronomic parameters, its incidence and severity, and the contents of stress biomarkers, secondary metabolites and photosynthetic pigments were determined. The results show that the impact of the pathogen on severity was about 60%; therefore, the impact on fruit yield was also negative (−31%). Moreover, the pathogen significantly impacted the content of stress biomarkers, and the positive control increased H2O2 (+23.9%), malondialdehyde (+41.7%) and proline (+54.8%). In contrast, UV-A radiation significantly decreased Fol severity (−35.5%), and prevented its negative effect on the tomato plant/fruit yield. In addition, the application of UV-A radiation decreased the contents of stress biomarkers (−10.4% O2•−, −22% H2O2, and −16% MDA), and increased the contents of secondary metabolites (+13.2% flavonoids, +35% anthocyanins) and photosynthetic pigments (+17% β-carotene, +12% yellow pigments, and +19.8% total chlorophyll) in the plants inoculated with the pathogen. From the results obtained, it can be concluded that the application of UV-A radiation is a good alternative means to control the attack of pathogens such as Fol on tomato plants, without adverse consequences for the environment or the crop.