The aims of this study were to investigate the effects of hesperidin, a citrus flavonoid, on human root dentin demineralization and collagen preservation, and compare it with chlorhexidine and grape seed extract. Specimens were assigned to different treatment groups: hesperidin, chlorhexidine and grape seed extract. Specimens were subjected to pH cycling by demineralization for 14 h, incubation in testing solutions for 2 h and remineralization in presence of bacterial-derived collagenase for 8 h, for 8 days. Calcium release was measured by means of an atomic absorption spectrophotometer, and degraded collagen matrix was investigated by hydroxyproline assay. Specimens were assessed longitudinally with transverse micro-radiography to investigate lesion depth and mineral loss. In hesperidin and grape seed extract groups, demineralization was reduced when the collagen matrix was preserved. The hesperidin group showed the lowest value in lesion depth and mineral loss, indicating that hesperidin inhibited demineralization and probably enhanced remineralization even under fluoride-free conditions.