In this study, the effect of co-culture Lactobacillus acidophilus ATCC 4356 and Lactobaacillus. plantarum ATCC 20552 on enzyme production (phytase, α-amylase, α-galactosidase and asparaginase (ASNase)), as well as acrylamide (AA) and 5-Hydroxymethylfurfural (HMF) levels in fermented bread, were determined. Based on the findings herein, the maximum levels of phytase (112.8–163 (U/mL), α-amylase (84.9–180.6 U/mL), α-galactosidase (2.33–6.72 U/mL), and asparaginase (5.23–9.14 U/mL) were calculated after 8 h of co-culture fermentation. The phytase and asparaginase activity of L. plantarum was higher than that of L. acidophilus while L. acidophilus showed higher α-amylase and α-galactosidase activity compared to L. plantarum. The phytic acid session caused an increase in the mineral content, thereby synergically affecting the rise in the amylase and asparaginase activity. The results also showed that acrylamide and HMF levels in breads fermented using co-culture L. acidophilus and L. plantarum + yeast was much lower (185.7 ± 1.1–34.6 ± 0.3 μg/kg d.b. and 12.3 ± 1.1 – <0.5 mg/kg d.b.) compared with those in the yeast-only fermented bread (334.9 ± 0.8–208.1 ± 0.7 μg/kg d. b. and 33.6 ± 1.2–20.5 ± 0.7 mg/kg d.b.), respectively. According to the treatment results, co-culture fermentation as along with time and temperature of proofing could be considered as potential factors to reduce AA and HMF content.