The objective of this study was to construct lactoferrin (LFA-LFC) chimeric peptide gelatin drug-loaded fiber film by tissue engineering strategy, and study its bacteriostatic effect on oral pathogens (especially Candida albicans) and its effect on biofilm. First of all, LFA-LFC chitosan nanoparticles were prepared firstly, and then fluconazole gelatin (LF/GH/F) film loaded with LFA-LFC was prepared by electrospinning. Scanning electron microscope (SEM), mechanical strength, drug release, cytotoxicity, and real-time PCR were used to test the properties of the synthesized materials. SEM showed that there was the reticular structure for fiber film before and after cross-linking. LF/GH/F film had no obvious cytotoxicity, with good biocompatibility and drug release; real-time PCR and antibacterial test showed that the LF/GH/F film had good antibacterial activity. LF/GH/F film has a good inhibitory effect on oral pathogens, and its mechanism is related to biofilm. The antibacterial experiments of nanofiber membrane in vitro and the effect of bacterial biofilm were carried out. The effect of LF/GH/F on oral microbial flora structure was studied by fluorescence quantitative pest techniques.