Background: Bioactive glass (BAG) remineralization is a promising method for dental hard tissue regeneration. The aim of this study was to evaluate the microhardness of incipient enamel-like lesions with or without preconditioning by air abrasion using polyacrylic acid (PAA)-BAG before application of NovaMin remineralizing agent. Materials and Methods: Forty extracted human molars were selected, sectioned mesiodistally obtaining buccal and lingual halves, and embedded in resin molds. Specimens were randomly assigned to four groups ( n = 10) according to the remineralization protocol: G1 (control, artificial saliva), G2 (preconditioning), G3 (NovaMin), and G4 (preconditioning and NovaMin). Enamel windows 4 mm × 4 mm were done on the buccal and lingual surfaces. Specimens were immersed in a daily renewed demineralizing solution to create white spot lesions. Remineralizing agents were applied according to the manufacturer's instructions, and specimens were stored in a daily renewed artificial saliva. Microhardness was assessed using Vickers hardness number (VHN) at baseline (positive control), after demineralization (negative control), and after 24-h and 1-month remineralization. Results: The preconditioning/NovaMin group after 1 month showed a statistically significant high VHN, with no statistically significant difference between it and the positive control. This was followed by the NovaMin group after 1 month, while the lowest VHN was found in the demineralized group, with no statistically significant difference between it and the preconditioning group, whether after 24 h or 1 month. Conclusions: Enamel preconditioning with PAA-BAG air abrasion play a major role in enhancement of remineralization when it is accompanied with NovaMin. Furthermore, an extended period of time had helped to attain more benefits from NovaMin remineralization. Clinical Significance: Enamel remineralization with NovaMin after conditioning by bioactive glass air abrasion, provides the patients with a fast and durable treatment of incipient enamel lesions, which would reduce the possibility of future progression of demineralization and caries occurrence.