This study aimed to analyze the enamel remineralization efficacy of a novel fluoridated bioactive glass (F-BG) toothpaste compared to a standard fluoride toothpaste. Seventy-two enamel blocks (N = 72) were divided into groups of twenty-four blocks according to the toothpaste exposure—group 1: brushed with distilled water, group 2: brushed with fluoride toothpaste (ColgateTM), and group 3: brushed with F-BG toothpaste (BioMinFTM). Pre-brushing, enamel blocks were demineralized using 6 wt.% citric acid (pH = 2.4). Tooth brushing was performed using a mixture of respective toothpaste and artificial saliva (AS), and each enamel block received 5000 linear strokes. The samples were assessed for surface micro-hardness (to estimate Vickers hardness number, VHN), surface roughness (Ra), and volume loss/gain using micro-computed tomography (micro-CT). The highest increase in the VHN was noticed for group 3 (117.81) followed by group 2 (61.13), and all the intragroup comparisons were statistically significant (p < 0.05). Demineralization increased the Ra values, and a decrease was observed post-remineralization for all the groups. The maximum Ra decrease was observed for group 3 (−223.2 nm) followed by group 2 (−55.6 nm), and all the intragroup comparisons were again statistically significant (p < 0.05). Micro-CT investigation revealed that the enamel volume decreased after demineralization and increased after remineralization among all groups. The F-BG toothpaste showed greater enamel surface micro-hardness (increased VHN), smoother surface (low roughness), and better volume restoration (remineralization) in comparison to the fluoride toothpaste.