This study was aimed to assess the effect of time and temperature on the extraction of antioxidant compounds from jabuticaba seeds (Myrciaria cauliflora cv. Sabará), to optimize the solvent proportion (water, ethyl alcohol, and propanone), and to characterize the extract according to the chemical composition, antioxidant, and antimicrobial properties. Proximal composition, total phenolic content (TPC), antioxidant, and antimicrobial activities were analyzed. The optimized solvent ratio of 60% water and 40% propanone provided a mean TPC of 8.65 g GAE/100 g seeds and the antioxidant activity toward 2,2-diphenyl-1-picrylhydrazyl (DPPH) was 82.79% ± 0.50%. Time and temperature parameters did not influence the yield of TPC. The gross seed extract was partially purified and both exhibited a high antioxidant activity and antimicrobial potential toward Gram-positive and Gram-negative bacteria. The purified jabuticaba seed lyophilized extract contained a higher (P < 0.05) TPC, o-diphenols, flavonols, and antioxidant activity measured by the DPPH assay and total reducing capacity as compared to the gross lyophilized extract. Electrospray ionization coupled with tandem mass spectrometry (ESI-MS/MS) data showed the presence of ellagitannins and ellagic acid in the extracts, which are probably the responsible for the antimicrobial and antioxidant activities.