The selectivity of sensory neurons for stimuli is often shaped by a balance between excitatory and inhibitory inputs, making this balance an effective target for regulation. In the inferior colliculus (IC), an auditory midbrain nucleus, the amplitude and selectivity of frequency response curves are altered by the neuromodulator serotonin, but the changes in excitatory-inhibitory balance that mediate this plasticity are not well-understood. Previous findings suggest that the presynaptic 5-HT1B receptor may act to decrease the release of GABA onto IC neurons. Here, in vivo extracellular recording and iontophoresis of the selective 5-HT1B agonist CP93129 were used to characterize inhibition within and surrounding frequency response curves using two-tone protocols to indirectly measure inhibition as a decrease in spikes relative to an excitatory tone alone. The 5-HT1B agonist attenuated such two-tone spike reduction in a varied pattern among neurons, suggesting that the function of 5-HT1B modulation also varies. The hypothesis that the 5-HT1B receptor reduces inhibition was tested by comparing the effects of CP93129 and the GABA_A antagonists bicuculline and gabazine in the same neurons. The effects of GABA_A antagonists on spike count, tuning bandwidth, two-tone ratio, and temporal response characteristics mimicked those of CP93129 across the neuron population. GABA_A antagonists also blocked or reduced the facilitation of evoked responses by CP93129. These results are all consistent with the reduction of GABA_A-mediated inhibition by 5-HT1B receptors in the IC, resulting in an increase in the level of evoked responses in some neurons, and a decrease in spectral selectivity in others.