Background <p>In clinical practice, microabrasion and remineralising agents, or their combinations, could be effective for white spot lesions (WSL) treatment. This study evaluates the effectiveness of four treatments, including two novel remineralising agents, nanohydroxyapatite (nanoHAP) and calcium glycerophosphate (CaGP) in combination with/without microabrasion on WSL.</p> Methods <p>Artificially created WSL on human enamels were randomly divided into nine groups according to treatments: (1) Group FV/fluoride varnish (Bifluoride 10); (2) Group NanoHAP/nanohydroxyapatite gel (Biodent); (3) Group CaGP/calcium glycerophosphate gel (SPC Kozmetik); (4) Group ICON/resin infiltration (ICON); (5) Group Micro/microabrasion (Opalustre); (6) Group Micro + FV/microabrasion + fluoride varnish; (7) Group Micro + NanoHAP/ microabrasion + nanohydroxyapatite gel; (8) Group Micro + CaGP/microabrasion + calcium-glycerophosphate gel; (9) Group Micro + ICON/microabrasion + resin infiltration. Samples were analysed using a Vickers microhardness (VHN) testing and two fluorescence-based methods (FluoreCam and DIAGNOdent Pen/DDPen) at baseline, after demineralisation and after treatment(<i>n</i> = 10). SEM/EDS was used for the evaluation of surface morphology and elemental analyses (<i>n</i> = 5). The data were statistically analysed (<i>p</i> &lt; 0.05).</p> Results <p>After treatments, Groups NanoHAP and Micro + NanoHAP showed significantly higher increases in VHN than Groups CaGP, Micro, ICON, Micro + ICON, and Micro + CaGP (<i>p</i> &lt; 0.05). According to the DDPen score, Group NanoHAP showed significantly higher remineralising potential than Groups CaGP, ICON, and Micro + ICON (<i>p</i> &lt; 0.05). According to FluoreCam data, a significantly higher remineralising effect was determined for Group Micro + NanoHAP than Group ICON (<i>p</i> &lt; 0.05). SEM analysis revealed that the deposition of a new mineralised layer was present for Groups NanoHAP and CaGP. EDS analysis detected that Groups NanoHAP and CaGP had similar Ca/P, Na, Cl, C, and O contents to Group FV (<i>p</i> &gt; 0.05). Regarding the presence of microabrasion, no significant differences in VHN, DDPen, FluoreCam, and EDS results were found for NanoHAP- and CaGP-containing groups (<i>p</i> &gt; 0.05).</p> Conclusions <p>Novel remineralising agents nanoHAP and CaGP could provide promising results with nanoHAP showing superior potential, whereas microabrasion before remineralising agents did not produce a significant additional effect on WSL.</p> Clinical relevance <p>NanoHAP is an effective alternative to fluoride for treating white spot lesions and may reduce the need for additional microabrasion procedures.</p>