The purpose of this in vitro study was to identify the secondary metabolites and to evaluate the antimicrobial activity, cytotoxicity, antioxidant capacity, and effect on metalloproteinases (MMPs) activity of two Brazilian propolis samples. The extracts of brown (BP) and green (GP) propolis were obtained by rotoevaporation. Phenolic compounds, flavonoids and coumarins were identified by colorimetric methods and determined by spectrophotometry. The minimum inhibitory concentration (MIC) of BP and GP against Candida albicans and Enterococcus faecalis was determined by broth microdilution. Cytotoxicity was assessed by MTT assay using L929 mice fibroblast cell line and supplemented DMEM culture medium. The antioxidant capacity was evaluated by DPPH test. The zymography assay was performed to evaluate the activity of BP and GP against MMP-2 and MMP-9. GP had a higher rate of coumarins and flavonoids, whereas BP had a higher content of phenolic compounds. GP and BP extracts showed antimicrobial activity against C. albicans and E. faecalis regardless of concentration, and presented low toxicity, except GP at 2.5 mg/mL, which reduced 40% of fibroblast cell metabolism. GP and PB extracts showed antioxidant capacity against the DPPH free radical at a concentration of 55.489 ± 1.512 μg/mL and 38.378 ± 0.735 μg/mL, respectively. Also, BP and GP showed an inhibitory effect against MMP-2 and MMP-9 from 1 to 5 mg/mL. The green and brown Brazilian propolis showed antimicrobial effect, low cytotoxicity, antioxidant capacity, and ability to inhibit the activity of MMP-2 and MMP-9, having potential to be used in endodontics as root canal irrigant.