The photosynthetic pigments are mainly responsible for absorbing the light intended to promote photosynthesis on the chloroplast of the leaves. Different studies have related the spectral response in the leaves of plants with the biotic stress generated by pathogens. In general, maximum differences in reflectance have been found in the range of 380–750 nm between plants subjected to biotic stress and healthy plants. In this study, it was possible to characterize and relate the spectral variance in leaves of S. lycopersicum infected with F. oxysporum with this physiological variation and pathogen concentration in tomato plants during the asymptomatic period of vascular wilt. Photosynthetic parameters derived from gaseous exchange analysis in the tomato leaves correlated related with four bands in the visible range (Vis). Additionally, five specific bands also present a high correlation with the increase in the concentration of F. oxysporum conidia measured at the root: 448–523 nm, 624–696 nm, 740–960 nm, 973–976 nm, and 992–995 nm. These wavelengths allowed a 100% correct classification of the plants inoculated with F. oxysporum from the plants subjected to hydric stress and the control plants in the asymptomatic period of the disease. The spectral response to biotic and abiotic stress in the measured Vis/NIR range can be explained by the general tendency to change the concentration of chlorophyll and carotene in tomato leaves. These studies also highlight the importance of the implementation of robust multivariate analysis over the multiple univariate analysis used in the applied biological sciences and specifically in the agricultural sciences. These results demonstrate that specific wavelength responses are due to physiological changes in plants subjected to stress, and can be used in indexes and algorithms applied to the early detection of diseases in plants on different pathosystems.