Summary Background Biologics targeting inflammatory mediators can achieve clinical improvements in hidradenitis suppurativa (HS). However, their clinical efficacy shows great interpatient variability in daily practice. Objectives To investigate the anti‐inflammatory potency of a selection of currently available biologics and prednisolone for the treatment of HS in an ex vivo skin culture system using lesional HS biopsies. Methods Lesional skin samples from 10 patients with HS and skin samples from five healthy controls were cultured ex vivo and exposed to prednisolone or biologics targeting tumour necrosis factor (TNF)‐α, interleukin (IL)‐17A, IL‐12/23p40 or CD20 (adalimumab, infliximab, secukinumab, ustekinumab and rituximab, respectively). Real‐time quantitative polymerase chain reaction and cytokine bead arrays were used to measure the inhibitory effect of the biologics on cytokines and antimicrobial peptides (AMPs). Results The relative mRNA expression of all tested cytokines and AMPs was significantly downregulated by all anti‐inflammatory agents (P < 0·001). The protein production of the proinflammatory cytokines TNF‐α, interferon γ, IL‐1β, IL‐6 and IL‐17A was significantly inhibited by adalimumab, infliximab, ustekinumab, prednisolone (all P < 0·001) and rituximab (P = 0·0071), but not by secukinumab (P = 0·0663). On both mRNA and protein levels, adalimumab, infliximab and prednisolone reduced the levels of a broader mix of individual cytokines than secukinumab, ustekinumab and rituximab. Moreover, a significant inhibitory effect on mRNA expression levels of inflammatory markers in healthy control skin was observed only for TNF‐α inhibitors (P < 0·001) and prednisolone (P = 0·0015). Conclusions This ex vivo study suggests that TNF‐α inhibitors and prednisolone are the most powerful inhibitors of proinflammatory cytokines and AMPs in HS lesional skin, which concurs with our clinical experience in patients with HS. What's already known about this topic? A key element of hidradenitis suppurativa (HS) is an aberrant immune response characterized by the overexpression of several proinflammatory cytokines and antimicrobial peptides in lesional skin. Biologics targeting inflammatory cytokines have the potential to improve HS disease activity. There is still need for efficacious drugs in the treatment of HS. What does this study add? We sought to quantify the anti‐inflammatory effects of currently available biologics in an ex vivo disease model. Adalimumab, infliximab, secukinumab, ustekinumab and rituximab in addition to prednisolone significantly inhibited a selected panel of proinflammatory cytokines and antimicrobial peptides in ex vivo HS lesional skin. Adalimumab, infliximab and prednisolone reduced the levels of a broader mix of individual cytokines than secukinumab, ustekinumab and rituximab. What is the translational message? The significant inhibition of important proinflammatory cytokines by tumour necrosis factor‐α inhibitors in HS correlates with their clinical efficacy. Our ex vivo skin culture system represents an adequate model for studies in search of novel candidate drugs for the treatment of HS and to personalize the treatment in specific patients. Linked Comment: Zouboulis. Br J Dermatol 2019; 181:244–246. Respond to this article