The recent global population explosion has increased people’s food demand. To meet this demand, huge amounts of nitrogen (N) fertilizer have been applied in the worldwide. However, ammonia (NH3) volatilization is one of the primary factors of N loss from soil after N application causing decrease crop N utilization efficiency and productivity. Incubation experiments were conducted on an acidic clayey soil with two different N sources (urea and anaerobic digestion effluent; ADE), two differently-produced biochars, and three biochar application rates (0%, 0.25%, and 1.0% w/w). Ammonia volatilization was lower from urea (14.0–23.5 mg N kg−1) and ADE (11.3–21.0 mg N kg−1) with biochar application than those without biochar (40.1 and 26.2 mg N kg−1 from urea and ADE alone, respectively). Biochar application significantly mitigated volatilization and reduction percentages for urea and ADE were 40%–64% and 18%–55%, respectively. 1.0% biochar application mitigated volatilization significantly compared to 0.25% application regardless of N source and biochar types. Possible mechanism for volatilization mitigation for urea and ADE were increased N immobilization by soil microorganisms and accelerated net nitrification rate due to increased soil nitrifying bacteria, respectively. Overall, our results clarified different mechanisms for N volatilization mitigation from different (inorganic vs. organic) N sources with biochar application.