Flavonoids, a group of plant secondary metabolites, are seen as chemicals for pharmaceutical, nutraceutical, and pesticidal applications. Due to their natural origin, anticipated low ecotoxicity and non-persistence in the environment, they are considered as benign substitutes for synthetic chemicals. However, data on ecotoxicity is still scarce. Therefore, this study aimed for a better understanding of the bioactivity of flavonoids and thus allowing a better evaluation of their potential as benign substitutes. The growth inhibition of the microalgae Raphidocelis subcapitata induced by 13 flavonoids, 10 flavonoid mixtures, and 4 flavonoid transformation products was determined according to the OECD guideline 201. EC50 values of flavonoids ranging from 0.7 to 22 mg/L were moderate in comparison to the synthetic algaecides terbutryn and diuron. Mixtures of flavonoids behaved in a dose-addition manner. Moreover, the fate of 11 selected flavonoids during the growth inhibition test was analyzed by analytical methods (HPLC-UV/vis and HPLC-HRMS). Concentration monitoring revealed fast but incomplete degradation of 8 flavonoids. Overall, 25 transformation products of the degrading flavonoids were identified including 5,7-dihydroxychromone. Comparison of structural features indicate that the combination of a methoxy group and a lacking hydroxy group at C3 decreased degradation rates. Growth inhibition of the 4 tested transformation products was weaker than of their respective parent flavonoids. Taken together, the results suggest low ecotoxicity of flavonoids and promote further research towards their utilization as benign substitutes.