Timbre perception and auditory grouping principles can provide a theoretical basis for aspects of orchestration. In Experiment 1, 36 excerpts contained two streams and 12 contained one stream as determined by music analysts. Streams—the perceptual connecting of successive events—comprised either single instruments or blended combinations of instruments from the same or different families. Musicians and nonmusicians rated the degree of segregation perceived in the excerpts. Heterogeneous instrument combinations between streams yielded greater segregation than did homogeneous ones. Experiment 2 presented the individual streams from each two-stream excerpt. Blend ratings on isolated individual streams from the two-stream excerpts did not predict global segregation between streams. In Experiment 3, Experiment 1 excerpts were reorchestrated with only string instruments to determine the relative contribution of timbre to segregation beyond other musical cues. Decreasing timbral differences reduced segregation ratings. Acoustic and score-based descriptors were extracted from the recordings and scores, respectively, to statistically quantify the factors involved in these effects. Instrument family, part crossing, consonance, spectral factors related to timbre, and onset synchrony all played a role, providing evidence of how timbral differences enhance segregation in orchestral music.