Endoanal ultrasound for the diagnosis of anal fistulas requires the injection of hydrogen peroxide, but it is often uncomfortable for the patient and possesses potential complications. Novel ultrasound contrast is currently available. We aimed to assess the efficacy and safety of sulfur hexafluoride as an ultrasound contrast agent for the diagnosis of perianal fistula by comparing it with those of 50% diluted hydrogen peroxide. Double-blind superiority study with 4 consecutive visits to perform an ultrasound without contrast, a hydrogen peroxide-enhanced ultrasound, a sulfur hexafluoride-enhanced ultrasound and a rectal exploration in the operating room (the gold standard). The ultrasound images were independently reviewed by three expert surgeon sonographers. This study was conducted at a single university hospital. Data from 176 patients were evaluated. Demographic and exploratory data and the ultrasound findings related to the location of the internal fistula orifice, description of the primary and secondary tracts, and presence of cavities and sphincter defects were analyzed. The complications occurring before and after the contrast agent administration and the presence of pain measured using a score were considered. Eighty-eight patients were included (men: 71.5%; mean age: 48.3 years).62.5% had a complex type and 83.7% had a transsphincteric type. Sulfur hexafluoride-enhanced ultrasounds demonstrated a higher interobserver agreement in determining the secondary tracts (κ= 0.604) and anal fistula height (κ=0.604) compared with other methods. Both hydrogen peroxide-enhanced ultrasound (90.91%) and sulfur hexafluoride-enhanced ultrasound (89.77%) detected the internal orifice more frequently than ultrasounds without contrast (62.5%) (p < 0.001),with no differences between contrast agents (p = 0.810). Sulfur hexafluoride-enhanced ultrasound were less painful than peroxide-enhanced ultrasound (p < 0.001). Most of the patients had transsphincteric anal fistulas. Sulfur hexafluoride proved comparable to hydrogen peroxide in evaluating fistulous tracts and identifying the internal orifice and additionally reduced significantly pain and discomfort. Furthermore, demonstrated a higher interobserver agreement in determining the secondary tracts and anal fistula height compared with other methods. See Video Abstract.