In the present study, a multiple approach was used to characterize Malpighia punicifolia extract and to evaluate its inhibitory activity against several meat spoilage bacteria. First, volatile fraction, vitamins and phenolic compounds of the extract obtained by supercritical fluid extraction were determined by GC‐MS and HPLC. Then, the antimicrobial action of the extract was in vitro evaluated against Pseudomonas putida DSMZ 291T, Pseudomonas fluorescens DSMZ 50009T, Pseudomonas fragi DSMZ 3456T, and Brochothrix thermosphacta DSMZ 20171T by the agar well diffusion assay and by the agar dilution test. Based on the results of the minimum inhibitory concentration (MIC) against the assayed bacteria, 4 different concentrations of the extract were used in a challenge test on water buffalo steaks stored for 21 d at 4 °C. Results of chemical analyses showed that M. punicifolia extract is characterized by the presence of several compounds, already described for their antimicrobial (phenolic acids, flavonones, and furanes) and antioxidant (ascorbic acid) properties. The in vitro detection of antimicrobial activities highlighted that the extract, used at 8% concentration, was able to inhibit all the target bacteria. Moreover, very low MIC values (up to 0.025%) were detected. In situ tests, performed on water buffalo steaks treated with the extract in the concentration range 0.025% to 0.05%, showed a strong inhibition of both intentionally inoculated bacteria and naturally occurring microorganisms. Positive results, in terms of color and odor, were also observed during the entire storage of steaks preserved with the extract.