In this study, the fibers of invasive species Agave americana L. and Ricinus communis L. were successfully used for the first time as new sources to produce cytocompatible and highly crystalline cellulose nanofibers. Cellulose nanofibers were obtained by two methods, based on either alkaline or acid hydrolysis. The morphology, chemical composition, and crystallinity of the obtained materials were characterized by scanning electron microscopy (SEM) together with energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. The crystallinity indexes (CIs) of the cellulose nanofibers extracted from A. americana and R. communis were very high (94.1％ and 92.7％, respectively). Biological studies evaluating the cytotoxic effects of the prepared cellulose nanofibers on human embryonic kidney 293T (HEK293T) cells were also performed. The nanofibers obtained using the two different extraction methods were all shown to be cytocompatible in the concentration range assayed (i. e., 0-500 μg/mL). Our results showed that the nanocellulose extracted from A. americana and R. communis fibers has high potential as a new renewable green source of highly crystalline cellulose-based cytocompatible nanomaterials for biomedical applications.