Background Aberrant kallikrein activity is observed in a number of inflammatory dermatoses. Up-regulation of kallikrein-5 (KLK5) activity leads to uncontrolled skin desquamation and cleavage of proteinase-activated receptor-2 (PAR2), causing the release of pro-inflammatory cytokines and disruption of epidermal barrier function. This study aimed to identify KLK5-specific small molecule inhibitors which can serve as the foundation of a novel therapeutic for inflammatory skin disorders. Methods Five chemical libraries (13,569 compounds total) were screened against recombinant KLK5 using a fluorogenic enzymatic assay. Secondary validation was performed on the top 22 primary hits. All hits were docked in the KLK5 crystal structure to rationalize their potential interactions with the protein. Results A naturally occurring compound derived from the wood of Caesalpinia sappan (Brazilin) was identified as a novel KLK5 inhibitor (IC50: 20 μM, Ki: 6.4 μM). Docking suggests that the phenolic moiety of Brazilin binds in the S1-pocket of KLK5 and forms a H-bond with S195 side chain. KLK14 was also found to be susceptible to inhibition by Brazilin with a calculated IC50 value of 14.6 μM. Conclusions Natural KLK5 small molecule inhibitors such as Brazilin, are ideal for topical skin disease drug design and remain a promising therapeutic for severe cases of inflammatory skin disorders. Optimized KLK inhibitors may have increased efficacy as therapeutics and warrant further investigation.