In recent decades, in order to replace traditional synthetic polymer composites, engineering research has focused on the development of new alternatives such as green biocomposites constituted by an eco-sustainable matrix reinforced by natural fibers. Such innovative biocomposites are divided into two different typologies: random short fiber biocomposites characterized by low mechanical strength, used for non-structural applications such as covering panels, etc., and high-performance biocomposites reinforced by long fibers that can be used for semi-structural and structural applications by replacing traditional materials such as metal (carbon steel and aluminum) or synthetic composites such as fiberglass. The present research work focuses on the high-performance biocomposites reinforced by optimized sisal fibers. In detail, in order to contribute to the extension of their application under fatigue loading, a systematic experimental fatigue test campaign has been accomplished by considering four different lay-up configurations (unidirectional, cross-ply, angle-ply and quasi-isotropic) with volume fraction Vf = 70%. The results analysis found that such laminates exhibit good fatigue performance, with fatigue ratios close to 0.5 for unidirectional and angle-ply (±7.5°) laminates. However, by passing from isotropic to unidirectional lay-up, the fatigue strength increases significantly by about four times; higher increases are revealed in terms of fatigue life. In terms of damage, it has been observed that, thanks to the high quality of the proposed laminates, in any case, the fatigue failure involves the fiber failure, although secondary debonding and delamination can occur, especially in orthotropic and cross-ply lay-up. The comparison with classical synthetic composites and other similar biocomposite has shown that in terms of fatigue ratio, the examined biocomposites exhibit performance comparable with the biocomposites reinforced by the more expensive flax and with common fiberglass. Finally, appropriate models, that can be advantageously used at the design stage, have also been proposed to predict the fatigue behavior of the laminates analyzed.