Interaction of myoglobin with cationic surfactants hexadecyltrimethylammonium bromide (HTAB), gemini surfactant 16-2-16 (dimethylene-1,2-bis(hexadecyldimethylammonium bromide)) and nonionic surfactant Mega 10 (N-decanoyl-N-methylglucamine) have been studied in phosphate buffer at pH 7.4 using surface tension, UV-visible, fluorescence, and circular dichroism spectroscopies and differential scanning calorimetry. With increasing concentration of HTAB, metal ion of the heme group changes its spin states; but in case of 16-2-16 and Mega 10, spin change does not occur. Fluorescence spectra clearly denote the unfolding process in HTAB media. With increasing HTAB concentration, alpha-helicity of myoglobin, decreases with the appearance of beta-sheet and random coil more rapidly than other two surfactants. Melting temperature of myoglobin is reduced drastically upon interaction with HTAB than their corresponding gemini and nonionic surfactants.