The interaction of phytopathogenic organisms and plants generates physiological and biochemical changes in the latter. However, the effects on the plants are rarely visible in the infection first stages. Novel optical techniques can help to improve the early detection of phytopathogenic organisms in tomato without the plant sacrifice. In this work, infrared spectroscopy and chemometric methods were used to determinate the effects of Fusarium oxysporum in tomato plants cultivated in pots, analyzing fully expanded leaves. Fusarium oxysporum was molecular identified and its pathogenicity was tested in vitro. Three plants treatments were evaluated for 55 days post infection in pots in greenhouse under semi-controlled conditions: control, water stress, and fungal inoculated (1 × 108 conidia/mL). Phenotypical results were followed twice a week for eight weeks; the phenotypical characteristics were very similar in almost all sampling times except in height, especially in the first 27 days post infection, after this time the height was similar in the three treatments. The stalk and root-dried matter analysis do not show statistical differences; however, the infrared results, acquired from the adaxial surface of leaves, show differences in peaks associated with salicylic acid, jasmonic acid, abscisic acid, and proline in the first 27 days post infection. The principal component analysis–linear discriminant analysis were used to distinguish subtle biochemical changes between the three treatments, facilitating the early detection of the pathogen and its monitoring over time.