The focus of caries research has switched to early identification and non-invasive treatment of carious lesions. This study aimed to evaluate and compare the remineralising potential of Ocimum (O.) basilicum varnish and fluoride varnish on initial enamel caries. The authenticated O. basilicum seeds were procured from a repository, and the extract was prepared using the Soxhlet method, which was vortexed with Indian Pharmaceutical (IP)-graded chemicals to obtain varnish. Extracted premolar tooth samples were divided into three groups of 33 each after demineralisation with a pH of 4.5 for 48 hours at 37°C. Each group was subjected to remineralisation twice daily with respective agents for 4 minutes for 30 consecutive days. Each sample was ground-sectioned through an enamel window. The lesion depth was measured using a light microscope (Leica™ DM2500) and ImageJ software. The data were evaluated using analysis of variance (ANOVA) and post hoc analysis. The mean (± SD) pre-treatment lesion depth across the groups ranged from 242.11 ± 26.144 μm to 352.66 ± 34.531 μm. The highest lesion depth recovery rate of 45.938% was recorded for the fluoride varnish group, followed by 36.015% in the O. basilicum varnish group, which was statistically significant by Tukey's post hoc analysis (p < 0.001). The gingival fibroblast cells were viable by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The O. basilicum varnish demonstrated a homogenous layer of mineral deposition. However, the remineralising efficacy was slightly lesser than that of the fluoride varnish. Hence, the novel O. basilicum-based remineralisation agent appears to have potential as a non-invasive alternative to topical fluorides in the therapy of early caries lesions.