This work describes a method for synthesis, as well as in vitro antiproliferative and antibacterial investigation of 3-methyl-9'-fluorenespiro-5-hydantoin. The structure of the substituted fluorenylspirohydantoin derivative was verified by UV-Vis, FT-IR, Raman, 1 H NMR and 13 C NMR spectroscopy, and by using a combination of 2D NMR experiments, which included 1 H-1 H COSY, HMQC and HMBC sequences. The geometry of the compound was optimized by the B3LYP density functional with 6-31G(d) basis set and the 1 H and 13 C NMR spectra were predicted with the HF/6-31G(d) calculations at the optimized geometry. The anticancer activity of the 3-methyl-9'-fluorenespiro-5-hydantoin was determined in suspension cell lines originating from tumors in humans (WERI-Rb-1). The cytotoxic effect was evaluated by WST-assay (Roche Applied Science). The antimicrobial effect of the compound against Gram-negative, Gram-positive bacteria and the yeast Candida albicans was investigated.