The genus Crotalaria comprises about 600 species that are distributed throughout the tropics and subtropical regions of the world; they are antagonistic to nematodes in sustainable crop production systems, and have also shown antimicrobial capacity. Chipilín (C. longirostrata), which belongs to this genus, is a wild plant that grows in the state of Chiapas (Mexico) and is traditionally is used as food. Its leaves also have medicinal properties and are used as hypnotics and narcotics; however, the plant has received little research attention to date. In the experimental part of this study, dried leaves were macerated by ethanol. The extract obtained was fractionated with ethyl ether, dichloromethane, ethyl acetate, 2-propanone, and water. The extracts were evaluated against three bacteria—namely, Escherichia coli (Ec), Citrobacter freundii (Cf), and Staphylococcus epidermidis (Se)—and three fungi—Fusarium oxysporum A. comiteca (FoC), Fusarium oxysporum A. tequilana (FoT), and Fusarium solani A. comiteca (FSC). During this preliminary study, a statistical analysis of the data showed that there is a significant difference between the control ciprofloxacin (antibacterial), the antifungal activity experiments (water was used as a negative control), and the fractions used. The aqueous fraction (WF) was the most active against FoC, FsC, and FoT (30.65, 20.61, and 27.36% at 96 h, respectively) and the ethyl ether fraction (EEF) was the most active against Se (26.62% at 48 h).