In this study, Anacardium occidentale (A. occidentale) nut skin waste (cashew nut skin waste) was used as a raw material to synthesize functionalized carbon nanodots (F-CNDs). A. occidentale biomass-derived F-CNDs were synthesized at a low temperature (200 °C) using a facile, economical hydrothermal method and subjected to XRD, FESEM, TEM, HRTEM, XPS, Raman Spectroscopy, ATR-FTIR, and Ultraviolet-visible (UV–vis) absorption and fluorescence spectroscopy to determine their structures, chemical compositions, and optical properties. The analysis revealed that dispersed, hydrophilic F-CNDs had a mean diameter of 2.5 nm. XPS and ATR-FTIR showed F-CNDs had a crystalline core and an amorphous surface decorated with –NH2, –COOH, and C=O. In addition, F-CNDs had a quantum yield of 15.5% and exhibited fluorescence with maximum emission at 406 nm when excited at 340 nm. Human colon cancer (HCT-116) cell assays showed that F-CNDs readily penetrated into the cells, had outstanding biocompatibility, high photostability, and minimal toxicity. An MTT assay showed that the viability of HCT-116 cells incubated for 24 h in the presence of F-CNDs (200 μg mL–1) exceeded 95%. Furthermore, when stimulated by filters of three different wavelengths (405, 488, and 555 nm) under a laser scanning confocal microscope, HCT-116 cells containing F-CNDs emitted blue, red, and green, respectively, which suggests F-CNDs might be useful in the biomedical field. Thus, we describe the production of a fluorescent nanoprobe from cashew nut waste potentially suitable for bioimaging applications.