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This study aimed to investigate the effects of spontaneous fermentation on physicochemical characteristics, bioactive compounds, and antioxidant activity of acerola and guava fruit industrial by-products. Viable cell counts of lactic acid bacterial (LAB) in acerola and guava by-products were ≥ 5.0 log CFU/mL from 24 h up to 120 h of fermentation. Fermented acerola and guava by-products had increased luminosity and decreased contrast. Contents of total soluble solids and pH decreased, and titrable acidity increased in acerola and guava by-products during fermentation. Ascorbic acid contents decreased in acerola by-product and increased in guava by-product during fermentation. Different phenolic compounds were found in acerola and guava by-products during fermentation. Fermented acerola and guava by-products had increased contents of total flavonoids, total phenolics, and antioxidant activity. The contents of total flavonoids and total phenolics positively correlated with antioxidant activity in fermented acerola and guava by-products. These results indicate that spontaneous fermentation could be a strategy to improve the contents of bioactive compounds and the antioxidant activity of acerola and guava by-products, adding value and functionalities to these agro-industrial residues.