Biologically derived carbon-based nanomaterials have been gaining attention in the recent years due to its various potential medical applications. In this study, fluorescent carbon nanoparticles (FCNPs) derived from an abundant alga, Cladophora vagabunda, is first reported. FCNPs with an average diameter of ~43 nm was synthesized via hydrothermal process, a simple and green method. The synthesized FCNPs exhibited absorbances in the ultraviolet to visible regions and emissions at 600 and 650 nm. Phytochemical screening and bioactivity tests were conducted to determine various bioactive compounds present in the FCNP surface and its antibacterial, antioxidant, and cytotoxic properties. Results showed that FCNPs from C. vagabunda contains tannin, saponin, flavonoids, and phenolic compounds which are known for their antimicrobial property as supported by the result of agar well diffusion method using a Gram-positive S. aureus and Gram-negative E. coli. Moreover, FCNPs shows inactive cytotoxicity using brine shrimp lethality test at ratio as high as 1:1 in 40% (v/v) ethanol. The results suggest the potential use of FCNPs from C. vagabunda as a fluorescent material with various biological activities.