BACKGROUND AND AIMS: Anti-tumor necrosis factor-alpha inhibitors (anti-TNFs) are the established treatment for perianal Crohn's disease (pCD), but relapse and non-response are common. Data on second- and third-line biologics are limited. We present the first direct comparison of second- and third-line biologics in pCD patients with active perianal disease previously treated with first-line anti-TNFs. METHODS: A multicenter retrospective cohort study included adult patients with pCD who failed first-line anti-TNF. The primary outcome was clinical perianal response, with secondary outcomes of radiological response (magnetic resonance imaging or transrectal ultrasound) and healing, and clinical remission. Propensity score matching (PSM) was used to adjust for baseline differences. RESULTS: A total of 486 pCD patients from 23 IBD centers were included, with 333/486 (68.5%) and 216/263 (82.1%) matched by PSM in the second and third-line treatment groups, respectively. In the second-line group, 62/78 (79.5%) of ustekinumab (UST)-treated patients achieved clinical perianal response, compared to 46/78 (58.9%) with vedolizumab (VDZ) (OR 4.47, 95% CI, 1.94-10.28, P < .001) and 38/78 (48.7%) with anti-TNFs (OR 5.29, 95% CI, 2.39-11.71, P < .001). In the third-line group, 38/49 (77.6%) of UST-treated patients achieved clinical perianal response, compared to 29/49 (59.2%) with VDZ (OR 9.96, 95% CI, 2.6-38.4, P < .001) and 27/49 (55.1%) with anti-TNFs (OR 12.03, 95% CI, 2.99-48.47, P < .001). UST-treated patients also had higher radiological response rates than VDZ (OR 3.28, 95% CI, 1.07-10.07, P = .038). CONCLUSION: In pCD patients failing anti-TNFs as first-line treatment, ustekinumab may be more effective than vedolizumab or another anti-TNF as second or third-line therapy.