Abstract Background The mechanism by which highly diluted and agitated solutions have their effect is still unknown, but the development in recent years of new methods identifying changes in water and solute dipole moments is providing insights into potential modes of action. Objective The objective of the current study was to compare the biological effects of Antimonium crudum (AC) previously obtained by our group and already described in the literature with now measurable physico-chemical effects on solvatochromic dyes. Methods Different dilutions of AC and succussed water have been characterized with respect to their effect on the visible spectra of the solvatochromic dyes methylene violet (MV), a pyridinium phenolate (ET33), and a dimethylamino naphthalenone (BDN) compared with in-vitro action against Leishmania amazonensis-infected macrophages. Results Dye responses varied according to the dye used and the level of AC dilution and results were found to corroborate previously published in-vivo and in-vitro effects of AC. In addition, a very significant enhancement in the absorbance increase of MV was seen using the supernatant from AC 200cH-treated cells (15%; p < 0.0001) over that seen with AC 200cH itself (4%; p = 0.034), suggesting the amplification of ultra-high dilution effects by biological systems. Furthermore, supernatants from AC-treated cells increased the range of dilutions of AC that were capable of producing effects on the spectra of MV. The effect of AC dilutions on dye ET33 was eliminated by a weak electric current passed through potency solutions. Conclusion The data confirm a correspondence between the biological effects of dilutions of AC in-vitro and physico-chemical effects on solvatochromic dyes as measured by changes in their visible spectra. Results also indicate high dilutions of AC are sensitive to exposure to electric currents and biological systems.