Haematoxylum campechianum is a medicinal plant employed as an astringent to purify the blood and to treat stomach problems such as diarrhea and dysentery. A bio-guided chemical fractionation of the methanolic extract obtained from this plant allowed for the isolation of five compounds: two chalcones known as sappanchalcone (1); 3-deoxysappanchalcone (2); three homoisoflavonoids known as hematoxylol A (3); 4-O-methylhematoxylol (4); and, hematoxin (5). The spasmolytic activity was determined in an in vitro model (electrically induced contractions of guinea pig ileum), and allowed to demonstrate that the methanolic extract (EC₅₀ = 62.11 ± 3.23) fractions HcF7 (EC₅₀ = 61.75 ± 3.55) and HcF9 (EC₅₀ = 125.5 ± 10.65) and compounds 1 (EC₅₀ = 16.06 ± 2.15) and 2 (EC₅₀ = 25.37 ± 3.47) of Haematoxylum campechianum present significant relaxing activity as compared to papaverine (EC₅₀ = 20.08 ± 2.0) as a positive control.