A microbial nucleoside phosphotransferase reaction was applied for the conversion of 5-fluorouridine (FUR) and 5-fluoro-2’-deoxyuridine (FUdR) into the corresponding fluorinated nucleotide analogs on a preparative scale. Under optimized conditions using washed cells of Serratia marcescens IFO 3046 and p-nitrophenyl phosphate as a phosphate donor, FUR and FUdR were converted to the phosphorylated form with conversion yields of 81.5% and 69.0%, respectively. Chemical and enzymatic analyses supported identification of the two fluorinated nucleotide products as 5-fluorouridine 5’-monophosphate (FUMP) and 5-fluoro-2-’deoxyuridine 5’-monophosphate (FdUMP). The phosphotransferase reaction product, FUMP, could be directly used for the successive synthesis of 5-fluorouridine diphosphate galactose using air-dried cells of Candida saitoana IFO 0768.