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To develop agar derivatives with good antibacterial activity and decreased gelling and melting temperatures, two agar–gentamycin conjugates with 9.20% and 12.68% gentamicin immobilized were fabricated by oxidation, Schiff base and reduction reaction, and characterized by a Fourier Transform Infrared Spectrometer, 1H nuclear magnetic resonance and an elemental analyzer. It was found that the modifications changed the intermolecular interactions, leading to decreased gelling and melting temperatures for the oxidized agar and slightly increased gelling and melting temperatures for agar–gentamycin conjugates. Further studies of antimicrobial properties showed that the two agar–gentamycin conjugates possessed good antibacterial activity, which was positively correlated with the dosage and the immobilization rate of gentamicin. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of agar–gentamycin conjugates with higher immobilization rates of gentamicin against Escherichia coli were 39.1 μg/mL and 156.2 μg/mL, respectively, and the MICs and MBCs against Staphylococcus aureus were 19.5 μg/mL and 78.1 μg/mL, respectively. A biofilm test indicated that certain concentrations of agar–gentamycin conjugate could effectively inhibit the biofilm formation of Escherichia coli and Staphylococcus aureus. In summary, agar–gentamycin conjugates possess good antibacterial activities and may be applied as a new kind of antibacterial material. [ABSTRACT FROM AUTHOR]