Posidonia oceanica balls (POB), a kind of seagrass, are a significant environmental issue since they are annually discharged onto beaches. Their current usefulness limits interest in their management and enhances the environmental problem. Therefore, in this research, the potential of this lignocellulosic biomass was studied from a holistic biorefinery point of view. To this end, an in-depth study was carried out to select the best pathway for the integral valorization of POBs. First, an autohydrolysis process was studied for the recovery of oligosaccharides. Then, a delignification stage was applied, where, in addition to studying different delignification methods, the influence of the autohydrolysis pre-treatment was also investigated. Finally, cellulose nanofibers (CNFs) were obtained through a chemo-mechanical treatment. The results showed that autohydrolysis not only improved the delignification process and its products, but also allowed the hemicelluloses to be valorized. Acetoformosolv delignification proved to be the most successful in terms of lignin and cellulose properties. However, alkaline delignification was able to extract the highest amount of lignin with low purity. CNFs were also successfully produced from bleached solids. Therefore, the potential of POB as a feedstock for a biorefinery was confirmed, and the pathway should be chosen according to the requirements of the desired end products.