Antimicrobial resistance and inflammation remain two of the most persistent global health problems. Due to the growing limitations of current treatment, new and safer therapeutic substances are increasingly needed. In this work, the biological potential of the ethyl acetate extract from Prosopis laevigata was investigated. Three fractions (R4, R7 and R9) were tested for antimicrobial activity against fourteen microorganisms and their anti-inflammatory properties were evaluated using a model of edema-induced TPA in mice. The fraction R9 showed the strongest antimicrobial effect with Minimum Inhibitory Concentration (MIC) values below 25 μg/mL against several clinically significant strains. The same fraction also reduced inflammation in vivo and reached inhibitory levels similar to those produced by indomethacin. These results suggest that P. laevigata contains active metabolites with dual biological activity, which underlines its importance as a possible natural source of anti-inflammatory and antimicrobial agents.