The aim of this in vitro study was to compare the efficacy of sodium-tri-metaphosphate-treated varnish containing eggshell and membrane powder and bioactive glass varnish with fluoride varnish in preventing erosion. Two windows were created on the buccal surface of 72 molars for the erosion cycle. One of the windows was treated with fluoride varnish [(FV, Metroberry, Imicryl), varnish containing STMP-treated eggshell and membrane powder (EPV, BioViera, Imicryl) and bioactive glass varnish (BAG, Polimo, Imicryl)] while the other window was used as control. Acidic syrup (Atarax) was applied 3 times a day and acidic drink (Coca Cola) 4 times a day for 5 days. The samples were analyzed by SEM (n = 1), ATR-FTIR (n = 6) and Vicker’s hardness (n = 5). Data were analyzed by one-way ANOVA and post hoc Tukey test. The protective effects of the varnishes were observed in the SEM images obtained. There was a difference between the FV and EPV groups in the 875 cm−1 v2 CO3−2 peak spectrum and microhardness values (p < 0.05). There was a difference between FV and FV-C in the carbonate v2 band in the acidic beverage demineralised varnish groups (p < 0.05). Other band areas and CO3−2 /PO4−3 ratios of the varnish areas against the erosion and control areas showed similar results (p > 0.05). Microhardness analyses showed that the BAG group demineralized with acidic syrup and the FV group demineralized with acidic drink were similar to the control group (p > 0.05). The varnishes tested in our study showed a similar protective effect against erosion as fluoride varnishes.