Introduction One hypothesis concerning the neural underpinnings of auditory streaming states that frequency tuning of tonotopically organized neurons in primary auditory fields in combination with physiological forward suppression leads to separable representations of high-frequency A and low-frequency B tones. The extent of spatial overlap between the tonotopic activations of A and B tones is thought to underlie the perceptual organization of streaming sequences into one coherent or two separate streams. The present study attempted to interfere with these mechanisms by transcranial direct current stimulation (tDCS) and to probe behavioral outcomes reflecting the perception of ABAB streaming sequences. Objectives We hypothesized that depending on the polarity, tDCS modulates the frequency selectivity and the degree of forward suppression in the neural populations activated by tones A and B. With depolarizing anodal stimulation, tonotopic activation should be broadened. Hyperpolarizing cathodal stimulation should narrow the spatial activation in auditory cortex (AC). As a perceptual effect, tDCS should therefore modulate the proportion of one-stream and two-stream perception whit anodal tDCS reducing two-stream and enhancing one-stream perception, whereas cathodal tDCS enhancing two-stream and reducing one-stream perception. Material and methods Twenty-two subjects were presented with ambiguous ABAB sequences of three different frequency separations and had to decide on their current percept. Before this psychophysical task, the subjects received sham, anodal, or cathodal tDCS over the left AC for 15 min. Each subject performed three consecutive sessions at 1-week intervals to avoid interference effects. In two out of three sessions, subjects were stimulated with anodal and cathodal direct current, respectively, and in one session they underwent a sham condition. Results We could confirm our hypothesis at the most ambiguous ΔF condition of 6 semitones. For anodal compared to sham and cathodal stimulation, we found a significant decrease in the proportion of two-stream and an increase in the proportion of one-stream perception. Conclusion The results demonstrate the feasibility of using tDCS to probe mechanisms underlying auditory streaming through the use of behavioral measures.