A simple, and fast method was developed for the simultaneous determination of five fungicides, namely thiram (THR), epoxiconazole (EPO), hexaconazole (HEX), tebuconazole (TEB), and diethofencarb (DIE), in different matrices by HPLC-UV. Parameters influencing the peak shape and resolution, such as the composition of mobile phase, pH and concentration of buffer solution, and column temperature, were examined and optimized. The proposed method was validated in terms of linearity, sensitivity, precision, and accuracy. Forced degradation studies were carried out for all analytes to demonstrate the specificity of the method and to evaluate the stability of analytes under different conditions. DNA interaction and DNA damage studies were conducted by HPLC and comet assay, respectively. All fungicides were found to bind DNA, except for DIE. While the binding coefficients for EPO, HEX, and TEB were of the order of 104, THR was found to interact more strongly with DNA with a binding coefficient of higher than 106. DIE did not induce DNA damage at any concentration tested. On the other hand, TEB, HEX, and EPO induced DNA damage up to 30 mu g/mL. THR showed cytotoxic effects at 20 and 30 mu g/ mL and caused significant DNA damage at lower concentrations.