Citrobacter freundii IFO 13545 produces a chitosan-like bioflocculant (BF) from acetate. The effects of other organic acids (citrate, fumarate, malate, glyoxylate, succinate, and oxaloacetate) as carbon sources on the growth and flocculation activity (FA) of the cultures were investigated. Citrate, fumarate, malate, succinate, or oxaloacetate supported the growth of IFO 13545 as a sole carbon source, but any FA was not detected during the growth on these organic acids. Glyoxylate did not serve as a carbon source. These organic acids were tested for a possibility as auxiliary carbon sources by adding each organic acid to a basal medium including 10 g l-1 acetate (AM). However, with the exception of malate, they all negatively affected FA at 1 g l-1 in AM. As a supply of amino acids, the effect of casamino acids on FA was also investigated. Unexpectedly, casamino acids considerably reduced FA at a concentration of 1 g l-1 in AM, although it improved the growth greatly. To determine which constituents caused FA reduction, the effects of individual amino acids were evaluated. Valine and tryptophan were found to strongly repress FA, even at low concentrations (≥10 mg l-1). Furthermore, to select complex organic nutrients suitable for BF production media, the effects exerted by yeast extract, peptone, and tryptone on FA were also investigated. Intriguingly, yeast extract and tryptone negatively affected FA, even at a concentration of 1 g l-1 , although peptone did not have significant effect at the same concentration. These results demonstrate that ubiquitous organic acids, amino acids, and complex organic nutrients can significantly affect the FA of IFO 13545 even at low concentrations.