Two types of antimicrobial glass fibers containing ZnO and CaO, with diameters ranging from tens of nanometers to 1 µm, were successfully fabricated by a laser spinning technique. The antimicrobial performance was corroborated according to ISO 20743:2013, by using gram-negative (Escherichia coli) and gram-positive (Streptococcus oralis, Streptococcus mutans and Staphylococcus aureus) bacteria, and yeast (Candida krusei) (more than 3 logs of reduction). The metabolic activity and endosomal system of eukaryotic cells were not altered by using eluents of CaO glass submicrometric fibers and ZnO fibers at 1 : 10 dilution as cellular media (viability rates over 70%). A dental material was functionalized by embedding ZnO nanofibers above the percolation threshold (20% wt), creating a three-dimensional (3D) fiber network that added an antimicrobial profile. This new ZnO glass fiber composite is proved non-cytotoxic and preserved the antimicrobial effect after immersion in human saliva. This is the first time that a fiber-reinforced liner with strong antimicrobial-activity has been created to prevent secondary caries. The potential of developing new fiber-reinforced composites (FRCs) with antimicrobial properties opens up an extensive field of dental applications where most important diseases have an infectious origin.