Nanomedicine has been used as a precise treatment for many diseases. The advantage of using nanodrugs is that they have more permeability and less toxicity to cells, which enhances the drug delivery system. Graphene is well known for its potential biological applications in drug, food, and pharma industries. This study aimed to assess the productivity and potentiality of nitrogen-doped graphene (NDG) and to evaluate their anticancer, antimicrobial, and biofilm inhibition activity. Nitrogen-doped graphene was synthesized by using a one-pot facile synthesis of NDG, wherein the NDG was prepared by the reduction of graphene oxide (GO) in the presence of hydrazine hydrate as a reducing agent, while ammonium hydroxide was used as a source of nitrogen on the surface of graphene. As-synthesized NDG was characterized by various characterization techniques such as UV-Vis, FT-IR, XRD, XPS, TEM, and N2 sorption studies analysis. Antimicrobial, anticancer, and biofilm inhibition assays were performed by standard protocols. N-doped graphene (NDG) showed better activity against Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtillis, Streptococcus pneumoniae, and Streptococcus mutans (p ≤ 0.05), whereas there was no activity against Gram-negative strains in Klebsiella pneumoniae and Pseudomonas aeruginosa. Biofilm inhibition was also improved with NDG compared to the standard ampicillin. NDG showed better results in both MCF-7 and Hela cell lines with IC50 of 27.15 µg/mL and 30.85 µg/mL, respectively. In conclusion, NDG has the best ability for use as a biomolecule, and research studies focusing on proteomics, metabolomics, and in vivo studies are needed to increase the impact of NDG in the drug and pharma industry.