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This work describes an efficient, simple, and green bioprocess for obtaining 5‐halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5‐fluoro‐2′‐deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L−1 min−1 (60%) of 5‐chloro‐2′‐deoxyuridine in 3 h. These halogenated pyrimidine 2′‐deoxynucleosides are used as antitumoral agents.

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