Benzodiazepines (BZs) and selective serotonin reuptake inhibitors (SSRIs) have both been approved for use in the treatment of anxiety disorders. Benzodiazepines offer immediate therapeutic benefits; however, tolerance and the potential for abuse may limit their long-term use. While little or no abuse potential is associated with SSRI use, therapeutic benefit is achieved only after several weeks of chronic treatment. During initial SSRI treatment, symptoms of anxiety may increase in some individuals and can lead to incomplete remission of anxiety symptoms or non-compliance. Evidence for the short-term use of BZs with long-term SSRI treatment is limited but some reports suggest faster symptom remission and/or greater global anxiolytic effect compared to SSRI treatment alone. The goal of the present research was to investigate the extent to which combinations of BZs and SSRIs interact to reduce behavioral and physiological measures of anxiety-like behavior and to investigate the mechanisms underlying these interactions. Acute treatment with SSRIs was shown to increase anxiogenic-like measures of behavior, with differences in dose-dependent decreases in locomotor activity associated with fluoxetine and citalopram, while a non-selective BZ (diazepam) produced acute anxiolytic-like effects with dose-dependent decreases in locomotor activity. Combinations of SSRI+BZ produced acute anxiolytic-like effects, attenuating both the anxiogenic-like effects associated with SSRI treatment and decreased locomotor activity associated with BZs. Investigation with GABAA subunit selective ligands indicate that α2, 3, and/or 5 subunit containing GABAA receptors are likely involved in the anxiolytic-like effects and decreases in locomotor activity, while the role of a1 subunit containing GABAA receptor is unclear, but play no role in decreased locomotor activity or anxiolytic-like effect of combined treatment. Investigation with selective serotonin (5-HT) receptor subtype ligands indicates that 5-HT1A and 5-HT2C receptors are not involved in the anxiogenic-like effects of acute SSRI treatment in this model. Acute treatment with anxiolytic-like dose of BZ, and anxiogenic-like dose of SSRI and anxiolytic-like doses of combined SSRI+BZ all result in significant increases in stress hormone release following administration. Altogether, these results suggest that acute, combined SSRI+BZ treatment may be useful in decreasing measures of anxiety-like behavior and minimizing decreases in locomotor activity and are implicated in a complex relationship with the hypothalamic pituitary axis.