Plantain seedlings availability in sub-Saharan countries are very weak to support a high productivity level required. The plantain cultivation faces problem of the seedling’s unavailability in quantity and quality for intensification of this crop. The amendment of PIF substrate production with natural products could be an alternative to this problem. This study objective is to evaluate the effect of vertical layers of Tithonia diversifolia flakes on the performance of PIF plantain seedlings in terms of vegetative growth and susceptibility to black Sigatoka disease (BSD) in nursery. The vertical layer of T. diversifolia flakes amendment was introduced in the propagator in the presence of the control without amendment in controlled and uncontrolled conditions for shoots, then seedlings generation, followed by the evaluation of the vegetative growth parameters, the inoculation of the leaves with Mycosphaerella fijiensis and the pool of biomarkers evaluation. The treatment increases the germination rate, the number of shoots, the height and the diameter of shoots, the area of leaves as well as the seedlings roots, but also protects the seedlings against BSD up to about 56% compared to the controls. It also enhances the accumulation of proteins, polyphenols content and enzymes such as peroxidase, polyphenoloxidase and glucanase. The dual role of the vertical layer of T. diversifolia flakes in seedling production as a biofertilizer and as a biopesticide was revealed in this research. Taking into account this bad herbs flakes as a tool for sustainable and green agriculture is an open door to poor peasants for their empowerment.