We studied the alkaloids in leaves and stems of three Buxus species (endemic B. colchica, introduced B. sempervirens and B. balearica) growing in Georgia, collected during flowering and fruiting stages from locations including near Tbilisi, Batum Botanical Garden, and Shaor Pass. Total alkaloids soluble in ether, CHCl3, and water were extracted using solvent methods, separated via buffer pH fractionation and column chromatography (Al2O3, SiO2). Pharmacological tests showed these alkaloids exhibited spasmolytic activity, with aqueous extract activity ordered as B. balearica > B. sempervirens > B. colchica. Alkaloids were identified by physicochemical (mp, [α]D) and spectral (UV, IR, PMR, MS) data, plus TLC comparison with authentic samples. Cycloprotobuxin-A was isolated from B. balearica; buxamine and cycloprotobuxin-A from B. sempervirens; cyclobuxin-D, pseudocyclobuxin-D, L-cycloprotobuxin-C, cycloprotobuxin-D, 3-N-dimethyl-C20N-methylaminocycloprotobuxin-C, pseudocyclobuxin-D (-)-N-oxide, buxamine, and buxaminol-G (latter two first reported in B. colchica) from B. colchica. Alkaloid content varied with growth stages: B. colchica had highest total alkaloids (1.88%) in leaves/stems during fruiting and highest buxamine (0.096%) during flowering; B. balearica showed highest total alkaloid yield (6% from leaves/stems during flowering); B. sempervirens had lowest buxamine. B. colchica from different locations had similar alkaloid profiles, suitable for pharmacological use. B. colchica should be collected during seed ripening for buxamine, and B. balearica was superior in alkaloid yield and activity.