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Introduction: Urease is an enzyme that catalyzes the hydrolysis of urea in the gastrointestinal and urinary systems, related with chronic bacterial infection. Piper is a basal angiosperm genus with ethnobotanical application. The aim of this study was to determine urease inhibitory activity of extracts from ten Guatemalan Piper species. Methods: Leaves from 7 species were collected in Suchitepequez (P. amalago, P. auritum, P. hispidum, P. jacquemontianum, P. oradendron, P. retalhuleuense, and P. umbellatum), and 3 in Alta Verapaz (P. psilorhachis, P. sempervirens, and P. variabile), shade-dried and milled. Dichloromethane (DCM) and methanol (MeOH) extracts were prepared by percolation and concentrated by rotavapor. The inhibitory effect was determined by enzymatic assays, qualitative by thin layer chromatography developed by phenol red; and quantitative by spectrophotometric kinetic method evaluated by microcolorimetry at 630 nm with phenol red. Results: Seventeen extracts out of 20 demonstrated anti-urease activity by TLC, showing one discoloration band with Rf 1.4-5.8; P. psilorhachis showed 2 discolorations bands at Rf 5.3 and 5.8, and the best activity (IC 50 MeOH 1.9 ± 0.08 μg/mL and DCM 2.1 ± 06 μg/mL), similar to positive control (hydrocyclohexanone IC 50 1.4 ± 0.05 µg/mL). Two other species, P. umbellatum (IC 50 MeOH 2.5 ± 0.06 µg/mL) and P. retalhuleunse (IC 50 MeOH 4.1 ± 0.7 µg/mL, DCM 4.8 ± 0.06 µg/mL) showed a moderate activity. Conclusion: Three species showed interesting anti-urease activity that deserves future studies by bioguided fractionation. Literature review demonstrated that this is the first report about the urease activity of these Piper species.