Periodontitis has been linked to obstructive sleep apnea (OSA). The stage III periodontitis is more prevalent in OSA severe and the oral microbiome changes due to an increase in periodontal pathogenic bacteria and C. albicans. Nanofiber drug delivery systems may be used for local antimicrobial therapy against microorganisms related to periodontitis and OSA. The aim of the present study was manufacture PCL/PLGA nanofibers incorporating an antifungal, Amphotericin B (AMB); an antimicrobial peptide, LfcinB (21–25)Pal (P61); and zinc oxide (ZnO), for the inhibition of in vitro polymicrobial biofilms. Nanofibers (Nf) were produced by electrospinning method: PCL/PLGA Nf loaded with: AMB (Nf 1), AMB-ZnO (Nf 2), P61 (Nf 3), P61–ZnO (Nf 4), AMB-P61 (Nf 5), AMB-P61-ZnO (Nf 6) and ZnO (Nf 7). Different methods were used to physically and chemically characterize the nanofibers. The cell viability of human periodontal ligament fibroblasts (HPdLF) treated with the nanofibers was higher than 50%, demonstrating the biocompatibility of the nanofibers obtained. The antimicrobial and anti-biofilm activities of the nanofibers were assessed using qualitative and quantitative analyses. Nine bacteria viability, Candida albicans viability, and biofilm formation were significantly inhibited by the Nf 6, and this effect increased with exposure time (72 h). The AMB-P61-ZnO-loaded PCL/PLGA nanofiber (Nf 6) may offer a promising strategy for treating localized cases of periodontitis in those with obstructive sleep apnea.