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Acmella oleracea is traditionally used by Amazonian folks to treat skin and mucous wounds, influenza, cough, toothache, bacterial and fungal infections. Its phytoconstituents, such as alkylamides, phenolic compounds, and terpenes, are reported to produce therapeutic effects, which justify the medicinal use of A. oleracea extracts. However, the scientific evidence supporting the application A. oleracea bioactive products for wound treatment of remains unexplored so far. This work aimed to characterize the phytochemical composition of methanolic extract of A. oleracea leaves (AOM) and to investigate their antioxidant, anti-inflammatory, antimicrobial and healing potential focusing on its application for wound healing. The dried leaves from A. oleracea submitted to static maceration in methanol for 40 days. The phytochemical constitution of AOM was analyzed based on the total phenolic dosage method and by UFLC-QTOF-MS analysis. Antioxidant activity was assessed by DPPH and NO scavenging activities, as well as MDA formation, evaluation of ROS levels, and phosphomolybdenum assays. In vitro anti-inflammatory activities were assessed by reduction of NO, IL-6, and TNF-α production and accumulation of LDs in peritoneal macrophages cells. Antimicrobial activity was evaluated by determining MIC and MBC/MFC values against P. aeruginosa , E. coli , S. epidermidis, S. aureu s and C. albicans , bacterial killing assay, and biofilm adhesion assessment. In vitro wound healing activity was determined by means of the scratch assay with L929 fibroblasts. Vanillic acid, quercetin, and seven other alkamides, including spilanthol, were detected in the UFLC-QTOF-MS spectrum of AOM. Regarding the biocompatibility, AOM did not induce cytotoxicity in L929 fibroblasts and murine macrophages. The strong anti-inflammatory activity was evidenced by the fact that AOM reduced the cellular production of inflammatory mediators IL-6, TNF-α, NO, and LDs in macrophages by 100%, 96.66 ± 1.95%, 99.21 ± 3.82%, and 67.51 ± 0.72%, respectively. The antioxidant effects were confirmed, since AOM showed IC 50 values of 44.50 ± 4.46 and 127.60 ± 14.42 μg/mL in the DPPH and NO radical inhibition assays, respectively. Additionally, AOM phosphomolybdenium reducing power was 63.56 ± 13.01 (RAA% of quercetin) and 104.01 ± 21.29 (RAA% of rutin). Finally, in the MDA quantification assay, AOM showed 63,69 ± 3.47% of lipid peroxidation inhibition. It was also observed that the production of ROS decreased by 69.03 ± 3.85%. The MIC values of AOM ranged from 1000 to 125 μg/mL. Adhesion of S. aureus, P. Aeruginosa, and mixed biofilms was significantly reduced by 44.71 ± 4.44%, 95.50 ± 6.37 %, and 51.83 ± 1.50%, respectively. AOM also significantly inhibited the growth of S. aureus (77.17 ± 1.50 %) and P. aeruginosa (62.36 ± 1.01%). Furthermore, AOM significantly enhanced the in vitro migration of L929 fibroblasts by 97.86 ± 0.82% compared to the control (P < 0.05). This study is the first to report total antioxidant capacity and intracellular LD reduction by AOM. The results clearly demonstrated that AOM exerts potent anti-inflammatory, antioxidant, antimicrobial, and wound healing effects, encouraging its further investigation and promising application in wound treatment. [Display omitted] • Acmella oleracea extract exhibited no cytotoxic effects on L929 fibroblasts and murine macrophages. • A. oleracea extract significantly reduced the production of inflammatory mediators (IL-6, TNF-α, NO) by macrophages. • A. oleracea extract M demonstrated antioxidant activity by scavenging free radicals and inhibiting lipid peroxidation. • A. oleracea extract exhibited antimicrobial antibiofilm activities against various bacterial and fungal strains. • A. oleracea extract significantly enhanced the migration of fibroblasts in vitro , indicating its potential for wound healing. [ABSTRACT FROM AUTHOR]